Transcriptomic and biochemical evidence for the role of lysine biosynthesis against linoleic acid hydroperoxide-induced stress in Saccharomyces cerevisiae.

Amino acid biosynthesis forms part of an integrated stress response against oxidants in Saccharomyces cerevisiae and higher eukaryotes. Here we show an essential protective role of the l-lysine biosynthesis pathway in response to the oxidative stress condition induced by the lipid oxidant-linoleic acid hydroperoxide (LoaOOH), by means of transcriptomic profiling and phenotypic analysis, and using the deletion mutant dal80∆ and lysine auxotroph lys1∆. A comprehensive up-regulation of lysine biosynthetic genes (LYS1, LYS2, LYS4, LYS9, LYS12, LYS20 and LYS21) was revealed in dal80Δ following the oxidant challenge. The lysine auxotroph (lys1∆) exhibited a significant decrease in growth compared with that of BY4743 upon exposure to LoaOOH, albeit with the sufficient provision of lysine in the medium. Furthermore, the growth of wild type BY4743 exposed to LoaOOH was also greatly reduced in lysine-deficient conditions, despite a full complement of lysine biosynthetic genes. Amino acid analysis of LoaOOH-treated yeast showed that the level of cellular lysine remained unchanged throughout oxidant challenge, suggesting that the induced lysine biosynthesis leads to a steady-state metabolism as compared to the untreated yeast cells. Together, these findings demonstrate that lysine availability and its biosynthesis pathway play an important role in protecting the cell from lipid peroxide-induced oxidative stress, which is directly related to understanding environmental stress and industrial yeast management in brewing, wine making and baking.

Transcriptomic insights into the molecular response of Saccharomyces cerevisiae to linoleic acid hydroperoxide.

Eukaryotic microorganisms are constantly challenged by reactive oxygen species derived endogenously or encountered in their environment. Such adversity is particularly applied to Saccharomyces cerevisiae under harsh industrial conditions. One of the major oxidants to challenge S. cerevisiae is linoleic acid hydroperoxide (LoaOOH). This study, which used genome-wide microarray analysis in conjunction with deletion mutant screening, uncovered the molecular pathways of S. cerevisiae that were altered by an arresting concentration of LoaOOH (75 µM). The oxidative stress response, iron homeostasis, detoxification through PDR transport and direct lipid ß-oxidation were evident through the induction of the genes encoding for peroxiredoxins (GPX2, TSA2), the NADPH:oxidoreductase (OYE3), iron uptake (FIT2, ARN2, FET3), PDR transporters (PDR5, PDR15, SNQ2) and ß-oxidation machinery (FAA2, POX1). Further, we discovered that Gpx3p, the dual redox sensor and peroxidase, is required for protection against LoaOOH, indicated by the sensitivity of gpx3Δ to a mild dose of LoaOOH (37.5 µM). Deletion of GPX3 conferred a greater sensitivity to LoaOOH than the loss of its signalling partner YAP1. Deletion of either of the iron homeostasis regulators AFT1 or AFT2 also resulted in sensitivity to LoaOOH. These novel findings for Gpx3p, Aft1p and Aft2p point to their distinct roles in response to the lipid peroxide. Finally, the expression of 89 previously uncharacterised genes was significantly altered against LoaOOH, which will contribute to their eventual annotation.

Prenatal programming of hepatocyte nuclear factor 4alpha in the rat: A key mechanism in the 'foetal origins of hyperglycaemia'?

AIMS/HYPOTHESIS: Prenatal glucocorticoid exposure causes lifelong hyperglycaemia in rat offspring, associated with permanently increased hepatic phosphoenolpyruvate carboxykinase 2 (PCK2), the rate-controlling enzyme of gluconeogenesis. To elucidate the mechanisms underlying the 'programming' of PCK2, this study examined the effect of prenatal dexamethasone treatment on expression of transcription factors that regulate Pck2. MATERIALS AND METHODS: Real-time RT-PCR and in situ hybridisation were used to measure and localise hepatic mRNA transcribed from the genes for PCK2, hepatocyte nuclear factor 4, alpha (HNF4A), transcription factor 1 (TCF1), CCAAT/enhancer binding protein, alpha (CEBPA), CEBPB, the glucocorticoid receptor (NR3C1) and peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (PPARGC1A) in foetal and adult offspring of dams treated with dexamethasone or vehicle during the last week of gestation. RESULTS: Prenatal dexamethasone exposure significantly elevated Hnf4a mRNA expression in foetal and adult liver. This resulted from increased expression of isoforms derived from the 'adult' (P1) Hnf4a promoter. In contrast, isoforms from the 'foetal' (P2) promoter were markedly suppressed by dexamethasone. Like Pck2, the increase in hepatic Hnf4a mRNA occurred exclusively in the periportal zone. Foetal Tcf1 expression was also increased by dexamethasone treatment, but this did not persist into adulthood. Prenatal dexamethasone did not affect the amounts of foetal and/or adult Cebpa, Cebpb, Nr3c1 or Ppargc1a mRNA. CONCLUSIONS/INTERPRETATION: Prenatal dexamethasone exposure caused a permanent increase in hepatic Hnf4a mRNA. This increase, which was associated with a premature switch from foetal to adult promoter predominance, was congruent with changes in Pck2 expression. These data suggest that HNF4A might mediate Pck2 overexpression and subsequent hyperglycaemia.

C/EBP regulates hepatic transcription of 11beta -hydroxysteroid dehydrogenase type 1. A novel mechanism for cross-talk between the C/EBP and glucocorticoid signaling pathways.

Glucocorticoid action within individual cells is potently modulated by 11beta-hydroxysteroid dehydrogenase (11beta-HSD), which, by interconverting active and inert glucocorticoids, determines steroid access to receptors. Type 1 11beta-HSD (11beta-HSD1) is highly expressed in liver where it regenerates glucocorticoids, thus amplifying their action and contributing to induction of glucocorticoid-responsive genes, most of which are also regulated by members of the C/EBP (CAAT/enhancer-binding protein) family of transcription factors. Here we demonstrate that C/EBPalpha is a potent activator of the 11beta-HSD1 gene in hepatoma cells and that mice deficient in C/EBPalpha have reduced hepatic 11beta-HSD1 expression. In contrast, C/EBPbeta is a relatively weak activator of 11beta-HSD1 transcription in hepatoma cells and attenuates C/EBPalpha induction, and mice that lack C/EBPbeta have increased hepatic 11beta-HSD1 mRNA. The 11beta-HSD1 promoter (between -812 and +76) contains 10 C/EBP binding sites, and mutation of the promoter proximal sites decreases the C/EBP inducibility of the promoter. One site encompasses the transcription start, and both C/EBPalpha and C/EBPbeta are present in complexes formed by liver nuclear proteins at this site. The regulation of 11beta-HSD1 expression, and hence intracellular glucocorticoid levels, by members of the C/EBP family provides a novel mechanism for cross-talk between the C/EBP family of transcription factors and the glucocorticoid signaling pathway.

5'-heterogeneity of glucocorticoid receptor messenger RNA is tissue specific: differential regulation of variant transcripts by early-life events.

Glucocorticoid receptor (GR) gene expression is regulated in a complex tissue-specific manner, notably by early-life environmental events that program tissue GR levels. We have identified and characterized several new rat GR mRNAs. All encode a common protein, but differ in their 5'-leader sequences as a consequence of alternate splicing of, potentially, 11 different exon 1 sequences. Most are located in a 3-kb CpG island, upstream of exon 2, that exhibits substantial promoter activity in transfected cells. Ribonuclease (RNase) protection analysis demonstrated significant levels of six alternate exons 1 in vivo in rat, with differences between liver, hippocampus, and thymus reflecting tissue-specific differences in promoter activity. Two of the alternate exons 1 (exons 1(6) and 1(10)) were expressed in all tissues examined, together present in 77-87% of total GR mRNA. The remaining GR transcripts contained tissue-specific alternate first exons. Importantly, tissue-specific first exon usage was altered by perinatal environmental manipulations. Postnatal handling, which permanently increases GR in the hippocampus, causing attenuation of stress responses, selectively elevated GR mRNA containing the hippocampus-specific exon 1(7). Prenatal glucocorticoid exposure, which increases hepatic GR expression and produces adult hyperglycemia, decreased the proportion of hepatic GR mRNA containing the predominant exon 1(10), suggesting an increase in a minor exon 1 variant. Such tissue specificity of promoter usage allows differential GR regulation and programming.

A novel nitric oxide scavenger decreases liver injury and improves survival after hemorrhagic shock.

We tested the ability of a nitric oxide (NO) scavenger to reduce tissue injury in a rodent model of hemorrhagic shock. Rats were hemorrhaged to a mean arterial blood pressure (MAP) of 40 mmHg and then resuscitated when either 30% of their shed blood had been returned (group 1) or after 100 min of continuous shock (group 2). Selected animals were treated with the NO scavenger NOX (30 mg. kg(-1). h(-1)) infused over 4 h. Hemorrhaged rats had a lower MAP after resuscitation compared with sham-shock control rats. NOX treatment significantly increased MAP after resuscitation from hemorrhage. Hemorrhagic shock also increased liver injury as reflected by elevated ornithine carbamoyltransferase (OCT) plasma levels, and NOX treatment significantly reduced OCT release. In addition, NOX was associated with significantly decreased hepatic neutrophil infiltration and improved 24-h survival (n = 8 of 9) compared with saline-treated shock animals (n = 3 of 9). These data suggest that excess NO mediates shock-induced tissue injury and that suppression of NO availability with NO scavengers may reduce the pathophysiological sequelae of severe hemorrhage.

A role of neutrophils in the down-regulation of IL-6 and CD14 following hemorrhagic shock.

Hemorrhagic shock (HS) followed by resuscitation has been shown to initiate a series of events, including local cytokine production and PMN accumulation. To determine whether PMN are involved in the regulation of IL-6 expression in the liver or lungs, IL-6 mRNA levels were measured in rats made neutropenic by vinblastine pretreatment prior to HS. IL-6 mRNA levels were determined at 4 or 24 h following resuscitation from shock. Vinblastine alone in normal rats or sham-treated rats had no effect at 4 or 24 h. Vinblastine pretreatment had no effect on the HS-induced increase in IL-6 mRNA at 4 h but dramatically increased levels in both liver and lung at 24 h. Peripheral PMN counts were reduced by 95% in all vinblastine-treated animals. Similar changes seen in CD14 mRNA expression indicate that these effects are not limited to IL-6. These data show that normal PMN levels are not needed for induction of IL-6 and CD14 in HS, and suggest that PMN accumulation down-regulates the expression of these genes.

G-CSF instillation into rat lungs mediates neutrophil recruitment, pulmonary edema, and hypoxia.

Activated neutrophils (PMN) have been implicated in the pathogenesis of adult respiratory distress syndrome (ARDS). Granulocyte colony-stimulating factor (G-CSF) is essential for PMN production and activation of PMN functions. We have recently shown that levels of G-CSF mRNA in a rat model of hemorrhagic shock correlated with severity of shock, PMN infiltration, pulmonary edema, and hypoxia. To determine whether increased tissue levels of G-CSF contribute to PMN recruitment and PMN-mediated injury, we instilled G-CSF into the lungs by intratracheal injection. Animals treated with G-CSF became hypoxic, hypocapnic, and alkalotic and demonstrated increased BAL fluid cellularity compared with control animals. The wet-to-dry ratio increased significantly after G-CSF instillation and peaked at 12 h. Histological examination of the lungs from G-CSF-treated rats revealed marked edema and increased PMN within the interstitium and alveoli. These results indicate that the presence of G-CSF alone in the lung can lead to recruitment of PMN, lung injury, and impaired pulmonary function, suggesting that local production of G-CSF may contribute to the development of lung damage and possibly ARDS in the setting of resuscitated hemorrhagic shock.

Cloning and production of antisera to human placental 11 beta-hydroxysteroid dehydrogenase type 2.

By inactivating potent glucocorticoid hormones (cortisol and corticosterone), 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) plays an important role in the placenta by controlling fetal exposure to maternal glucocorticoids, and in aldosterone target tissues by controlling ligand access to co-localized glucocorticoid and mineralocorticoid receptors. Amino acid sequence from homogeneous human placental 11 beta-HSD2 was used to isolate a 1897 bp cDNA encoding this enzyme (predicted M(r) 44126; predicted pI 9.9). Transfection into mammalian (CHO) cells produces 11 beta-HSD2 activity which is NAD(+)-dependent, is without reductase activity, avidly metabolizes glucocorticoids (Km values for corticosterone, cortisol and dexamethasone of 12.4 +/- 1.5, 43.9 +/- 8.5 and 119 +/- 15 nM respectively) and is inhibited by glycyrrhetinic acid and carbenoxolone (IC50 values 10-20 nM). Rabbit antisera recognizing 11 beta-HSD2 have been raised to an 11 beta-HSD2-(370--383)-peptide-carrier conjugate. Recombinant 11 beta-HSD2, like native human placental 11 beta-HSD2, is detectable with affinity labelling and anti-11 beta-HSD2 antisera, and appears to require little post-translational processing for activity. 11 beta-HSD2 mRNA (approximately 1.9 kb transcript) is expressed in placenta, aldosterone target tissues (kidney, parotid, colon and skin) and pancreas. In situ hybridization and immunohistochemistry localize abundant 11 beta-HSD2 expression to the distal nephron in human adult kidney and to the trophoblast in the placenta. 11 beta-HSD2 transcripts are expressed in fetal kidney (but not lung, liver or brain) at 21-26 weeks, suggesting that an 11 beta-HSD2 distribution resembling that in the adult is established by this stage in human development.

Cloning, sequencing and tissue-distribution of mouse 11 beta-hydroxysteroid dehydrogenase-1 cDNA.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) reversibly converts physiological glucocorticoids (cortisol, corticosterone) to inactive 11-dehydro forms, and thus controls glucocorticoid access to receptors in a variety of tissues. We have cloned a cDNA encoding 'liver-type' 11 beta-HSD (11 beta-HSD1) from the mouse using PCR, and have determined its nucleotide sequence. Mouse 11 beta-HSD1 cDNA showed 91% identity to rat 11 beta-HSD1 cDNA. There was 87% amino acid identity with rat 11 beta-HSD1 with conservation of the putative cofactor and substrate binding domains. Northern blot analysis of mouse tissues demonstrated abundant 11 beta-HSD1 message in the liver, kidney and lung, with lower expression in brain subregions and gonads. 11 beta-HSD1 mRNA was below the level of detection in the murine colon. 11 beta-HSD1 mRNA levels in kidney was higher in males than in females, but in contrast to the rat, there was no sexual dimorphism in the mouse liver. Although males and females showed different mRNA levels in the kidney, there was no sex difference in 11 beta-HSD enzyme activity. Thus, despite the high inter-species conservation of 11 beta-HSD1, there are clear species and tissue-specific differences in its expression.

The rat preprotachykinin-A promoter is regulated in PC12 cells by the synergistic action of multiple stimuli.

We demonstrate in PC12 cells that although nerve growth factor, forskolin or potassium-evoked depolarisation independently induced minimal or no expression from the rat preprotachykinin-A gene (rPPT) promoter linked to a reporter gene, exposure of the cells to various combinations of these stimuli specifically activated the rPPT promoter in transient transfection assays.

The sequence of 5' flanking DNA from the rat preprotachykinin gene; analysis of putative transcription factor binding sites.

The rat preprotachykinin A (rPPT-A) gene is expressed in restricted populations of neurons and endocrine cells, including a subset of dorsal root ganglion (DRG) neurons. As part of a study to investigate the DNA sequences responsible for tissue-specific expression of the gene, we have determined the sequence of the 5' flanking DNA to 3350 bp upstream of the transcription start site. The sequenced region encodes a number of putative transcription factor binding sites which may play important roles in the regulation of rPPT-A gene transcription.

5'-flanking sequences from the rat preprotachykinin gene direct high-level expression of a reporter gene in adult rat sensory neurons transfected in culture by microinjection.

The rat preprotachykinin-A gene, which encodes substance P, is expressed in response to nerve growth factor in a subpopulation of dorsal root ganglion sensory neurons. To investigate mechanisms regulating preprotachykinin-A transcription, we transfected adult rat sensory neurons in culture by microinjection of plasmids containing genomic DNA sequences linked to a lacZ (beta-galactosidase) reporter gene. Expression of beta-galactosidase was seen in 10-15% of neurons receiving injections of prPPT-betaGAL1, which contained the preprotachykinin transcription start site and 3356 bp of 5'-flanking DNA. Deletion analysis showed that expression was directed by 865 bp lying immediately upstream of the transcription start site. Extension of the prPPT-betaGAL1 sequence to include the first intron of preprotachykinin increased beta-galactosidase two- to threefold. Functional promoter and enhancer sequences from the rat prolactin gene failed to direct expression in sensory neurons, indicating neuronal selectivity for preprotachykinin sequences. Expression of prPPT-betaGAL1, measured relative to a construct containing the Rous sarcoma virus promoter, was approximately fivefold higher in neurons than in nonneuronal cells. This suggests selectivity by preprotachykinin 5'-flanking sequences for neuronal expression. However, prPPT-betaGAL1 expression was not restricted to the neuronal subpopulation containing immunoreactive substance P nor was it dependent upon nerve growth factor. Therefore, it does not share all the characteristics of endogenous preprotachykinin expression implying the need for additional regulatory sequences or the involvement of post-transcriptional regulation. Our results show that transfection of differentiated neurons in culture by microinjection has considerable potential in studies of neuron-specific gene expression.

Bacterial adherence to plain and tobramycin-laden polymethylmethacrylate beads.

Antibiotic-impregnated polymethylmethacrylate (PMMA) bead chains are in current clinical use for prophylaxis and management of osteomyelitis. The in vivo interaction between PMMA beads and an experimentally infected wound is examined. Two modes of bacterial adherence to plain PMMA beads are demonstrated. In this report, tobramycin-sensitive bacteria did not attach to tobramycin-laden beads. Therefore, implanted PMMA beads should contain an antibiotic to which the infecting bacteria is sensitive.

Regulation of pituitary alpha-subunit, beta-luteinizing hormone and prolactin messenger ribonucleic acid by gonadotropin-releasing hormone and estradiol in hypogonadal mice.

We have investigated the effects of three different GnRH injection regimens and the effects of estradiol benzoate (EB) on expression of the common alpha-subunit, beta-LH, and PRL genes in male and female hpg mice. GnRH was injected once daily (100 ng), every 2 h (100 ng) or every 30 min (25 ng), and EB (10 micrograms) was injected once daily. The effects of continuous exposure to the superactive agonist D-Trp6-GnRH released from microcapsules were also studied. Northern blot analysis showed that administration of GnRH increased alpha-subunit mRNA levels 2- to 10-fold in male and female hpg but not normal mice and had no significant effect on beta-LH or beta-TSH mRNA levels. The greatest increase in alpha-mRNA occurred when 100 ng GnRH were injected every 2 h and could be detected within 6 h of the first GnRH injection. More frequent injections (25 ng every 30 min) were less effective in increasing alpha-mRNA, as was prolonged exposure to the D-Trp6-GnRH superagonist. The increase in alpha-mRNA was associated with an increase in pituitary FSH content of similar magnitude. Continuous exposure of the pituitary gland to D-Trp6-GnRH (approximately 1500 ng/day) resulted in a smaller (2-fold) increase in alpha-mRNA and pituitary FSH content, suggesting that desensitization had occurred. EB had little effect on beta-LH mRNA and did not alter alpha-mRNA levels or affect the increase in alpha-mRNA caused by GnRH. Injection of GnRH every 2 h increased pituitary PRL mRNA levels in female but not male hpg mice, probably due to an indirect effect resulting from increased estrogen secretion. We conclude that GnRH administration to hpg mice significantly increases alpha-subunit but not beta-LH mRNA levels and that maximal effects occur with 100 ng GnRH injections every 2 h. Although EB does have direct effects upon pituitary gonadotropin content in hpg mice, the absence of significant changes in alpha- and beta-LH mRNA suggests that these effects may be largely posttranscriptional.

The priming effect of LH-releasing hormone: effects of cold and involvement of new protein synthesis.

The possible involvement of protein synthesis in the priming effect of LH-releasing hormone (LHRH) has been investigated in vitro using hemipituitary glands from pro-oestrous rats. Cycloheximide (7.1 mumol/l) blocked the priming effect of LHRH (elicited by 8.5 nmol LHRH/l) and protein synthesis (assessed by gel electrophoresis of 35S-labelled pituitary proteins). Pituitary glands were also incubated at 0-1 degrees C followed by incubation at 37 degrees C. While incubation at 0 degrees C for either 1 or 2 h reduced LH release and blocked protein synthesis, the LH response to LHRH in a subsequent 1-h incubation at 37 degrees C was similar to that during the corresponding period in pituitary glands incubated throughout at 37 degrees C. Incubation with medium alone at 0 degrees C during the first hour followed by incubation with LHRH at 37 degrees C during the second hour resulted in an LH response to LHRH which was similar to that in glands incubated with LHRH for 2 successive hours at 37 degrees C. Two-dimensional gel electrophoresis showed that LHRH priming was associated with the synthesis of a new protein of approximately 69 000 molecular weight and with changes in the isoelectric point of two other high molecular weight proteins. These results suggest that the priming effect of LHRH involves the synthesis of a new protein as well as post-translational changes (possibly phosphorylation) in two other proteins and that exposure to cold may prime the pituitary gland to LHRH possibly by stimulating intracellular Ca2+ release and/or protein phosphorylation.