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Bacillus subtilis forms dormant spores upon nutrient depletion. Under favorable environmental conditions, the spore breaks its dormancy and resumes growth in a process called spore germination and outgrowth. To elucidate the physiological processes that occur during the transition of the dormant spore to an actively growing vegetative cell, we studied this process in a time-dependent manner by a combination of microscopy, analysis of extracellular metabolites, and a genome-wide analysis of transcription. The results indicate the presence of abundant levels of late sporulation transcripts in dormant spores. In addition, the results suggest the existence of a complex and well-regulated spore outgrowth program, involving the temporal expression of at least 30% of the B. subtilis genome. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The aim of the work described in this report is to develop and characterize a cell-based androgen reporter assay. For this purpose, the androgen receptor (AR) expressing human breast cancer cell line T47D was stably transfected with a luciferase gene under transcriptional control of the PB-ARE-2 androgen response element. The application of this cell line in an endogenous Androgen Receptor-mediated LUciferase eXpression assay (AR-LUX) was validated. An EC50 value of 86 pM was determined for the standard androgen R1881 with a detection limit of 46 pM. Other androgens like dihydrotestosterone, 17β-trenbolone, and bolasterone also induced luciferase expression, while anti-androgens suppressed these responses. As expected, AR-mediated responses were also elicited by high concentrations of the steroids progesterone, 17β-estradiol, d-aldosterone, and dexamethasone, with observed EC50 values 10 to 350,000 times higher than that for R1881. A unique feature of the AR-LUX assay is that effects on modulation of active endogenous AR-levels are reliably reflected in the luciferase induction response, as exemplified by vitamin D, all-trans-retinoic acid, epigallocatechin gallate, and forskolin. This feature is especially useful when assessing complex mixtures, e.g., environmental samples or natural compound libraries. From these data it is concluded that the AR-LUX assay is a reliable in vitro test system for the detection and quantification of AR-mediated biological effects. The 96-well plate format makes the assay particularly suitable for high-throughput screening.Chemicals/CAS: Aldosterone, 52-39-1; Androgens; Dexamethasone, 50-02-2; Estradiol, 50-28-2; Luciferases, EC 1.13.12.-; Metribolone, 965-93-5; Receptors, Androgen

Tissue-type plasminogen activator (t-PA) gene expression in human endothelial cells and HeLa cells is stimulated by the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) at the level of transcription. To study the mechanism of transcriptional regulation, we have characterized a segment of the t-PA gene extending from -135 to +100 by in vivo footprinting analysis [dimethyl sulphate (DMS) method] and gel mobility shift assay. In vivo footprinting analysis revealed changes in cleavage pattern in five distinct promoter elements in both endothelial cells and HeLa cells, including a PMA-responsive element (TRE), a CTF/NF-1 binding site and three CC-boxes, and an altered cleavage pattern of the TRE and CTF/NF-1 element after PMA treatment of HeLa cells. Although endothelial cells and Hela cells differed in the exact G residues protected by nuclear proteins, in vitro bandshift analysis showed that nuclear protein binding to the t-PA promoter was qualitatively and quantitatively very similar in both cell types, except for the TRE. Protein binding to the TRE under non-stimulated conditions was much higher in human endothelial cells than in HeLa cells, and this TRE-bound protein showed a lower dissociation rate in the endothelial cells than in HeLa cells. In endothelial cells, the proteins bound to the TRE consisted mainly of the AP-1 family members JunD and Fra-2, while in HeLa cells predominantly JunD, FosB and Fra-2 were bound. The proteins bound to the other protected promoter elements were identified as SP-1 (GC-box II and III) and CTF/NF-1 (CTF/NF-1 binding site). After PMA treatment of the cells, AP-1 and SP-1 binding was increased two-fold in endothelial cell nuclear extracts and > 20-fold in HeLa nuclear extracts. In the endothelial cells, all Jun and Fos forms (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1 and Fra-2) were part of the AP-1 complex after PMA induction. In HeLa cells, the complex consisted predominantly of c-Jun and the Fos family members FosB and Fra-2. In the light of previous studies involving mutational analysis of the human and murine t-PA promoter our results underline an important role of the five identified promoter regions in basal and PMA-stimulated t-PA gene expression in intact human endothelial cells and HeLa cells. The small differences in DMS protection pattern and differences in the individual AP-1 components bound in endothelial cells and HeLa cells point to subtle cell-type specific differences in t-PA gene regulation. Chemicals/CAS: CCAAT-Enhancer-Binding Proteins; CTF-1 transcription factor; DNA Primers; DNA-Binding Proteins; Endopeptidases, EC 3.4.-; NFI Transcription Factors; Nuclear Proteins; Oncogene Proteins; Oncogene Proteins, Fusion; Tetradecanoylphorbol Acetate, 16561-29-8; Tissue Plasminogen Activator, EC 3.4.21.68; Transcription Factors; USP6 protein, human, EC 3.4.99.-

The barley lipoxygenase 1 (Lox1) gene encodes a protein expressed in embryos during grain development and germination and in leaves after methyl-jasmonate (MeJA) treatment. Transient gene expression assays in germinating barley embryos were used to identify cis-regulatory elements involved in the embryo-specific expression of the Lox1 gene. Analysis of transcriptional or translational fusions between Lox1 5' upstream sequences and the gusA reporter gene indicated that the 5'-untranslated leader sequence was involved in embryo-specific expression. Replacement of the leader sequence from the aleurone-specific Chi26 gene with the Lox1 leader sequence resulted in a chimeric gene expressed at high levels in embryo as well as in aleurone cells. Insertion of the Lox1 leader sequence between the 35S minimum promoter (A domain -90/+8) and the gusA reporter gene greatly enhanced promoter activity in a tissue-specific manner. Deletion/replacement analysis of the Lox1 leader sequence, combined with transient expression in germinating embryos and in vitro transcription/translation assays, suggests that the Lox1 leader sequence contains cis-elements regulating qualitative (tissue-specific) and quantitative gene expression.

Health is influenced by interplay of molecular, physiological and environmental factors. To effectively maintain health and prevent disease, health-relevant relations need to be understood at multiple levels of biological complexity. Network-based methods provide a powerful platform for integration and mining of data and knowledge characterizing different aspects of health. Previously, we have reported physiological and gene expression changes associated with adaptation of murine epididymal white adipose tissue (eWAT) to 5 days and 12 weeks of high-fat diet (HFD) and low-fat diet feeding (Voigt et al. in Mol Nutr Food Res 57:1423–1434, 2013. doi:10.1002/mnfr.201200671). In the current study, we apply network analysis on this dataset to comprehensively characterize mechanisms driving the short- and long-term adaptation of eWAT to HFD across multiple levels of complexity. We built a three-layered interaction network comprising enriched biological processes, their transcriptional regulators and associated changes in physiological parameters. The multi-layered network model reveals that early eWAT adaptation to HFD feeding involves major changes at a molecular level, including activation of TGF-β signalling pathway, immune and stress response and downregulation of mitochondrial functioning. Upon prolonged HFD intake, initial transcriptional response tails off, mitochondrial functioning is even further diminished, and in turn the relation between eWAT gene expression and physiological changes becomes more prominent. In particular, eWAT weight and total energy intake negatively correlate with cellular respiration process, revealing mitochondrial dysfunction as a hallmark of late eWAT adaptation to HFD. Apart from global understanding of the time-resolved adaptation to HFD, the multi-layered network model allows several novel mechanistic hypotheses to emerge: (1) early activation of TGF-β signalling as a trigger for structural and morphological changes in mitochondrial organization in eWAT, (2) modulation of cellular respiration as an intervention strategy to effectively deal with excess dietary fat and (3) discovery of putative intervention targets, such those in pathways related to appetite control. In conclusion, the generated network model comprehensively characterizes eWAT adaptation to high-fat diet, spanning from global aspects to mechanistic details. Being open to further exploration by the research community, it provides a resource of health-relevant interactions ready to be used in a broad range of research applications. © 2015, The Author(s).

Evidence from in vivo studies indicates that the bile acid pool and bile acid excretion are increased in humans with diabetes mellitus and in experimental diabetic animals, and that both parameters return to normal levels after administration of insulin. To investigate the biochemical background of these changes, the effects of insulin on bile acid synthesis and cholesterol 7α-hydroxylase and sterel 27-hydroxylase, two key enzymes in routing of cholesterol toward bile acids, were studied in cultured rat hepatocytes. Mass production of bile acids was dose dependently diminished, showing significant reduction (-33% to -53%) at physiological concentrations of the hormone (1.4 to 14 nmol/L) and a maximal decrease at 140 nmol/L (- 65%). The decrease of bile acid synthesis correlated well with the suppression of cholesterol 7α-hydroxylase and sterol 27-hydroxylase activity. The enzyme activity for cholesterol 7α-hydroxylase, examined in more detail, was dose dependently diminished on incubation of hepatocytes with various concentrations of insulin, reaching maximal reduction at 14 nmol/L of insulin. Maximal decrease of the enzyme activity was seen after 8 hours of incubation (-70%). Insulin strongly reduced the rise in cholesterol 7α-hydroxylase activity induced by incubation with dexamethasone. Sterol 27- hydroxylase activity was inhibited up to -58% after 24 hours of incubation with 140 nmol/L insulin. To study the mechanism of suppression of cholesterol 7α-hydroxylase and sterol 27-hydroxylase activity, the effects of insulin on their respective levels of messenger RNA (mRNA) and gene transcription were assessed. The decrease in enzyme activities could be explained by a concomitant reduction in the cholesterol 7α-hydroxylase (-76%) and sterol 27-hydroxylase (-62%) mRNA level. Transcriptional activity, as assessed by nuclear runoff assays, was decreased to the same extent, i.e., -60% for cholesterol 7α-hydroxylase and -75% for sterol 27-hydroxylase. Transient expression experiments using a construct containing the proximal 348 basepairs of the cholesterol 7α-hydroxylase promoter fused to the chloramphenicol acetyltransferase (CAT) gene (-348Rcat) showed a significant reduction of transcriptional activity (-64%) with insulin, indicating that a sequence important for an insulin-induced transcriptional response is located within the first 348 basepairs, preceding the transcription start of the cholesterol 7α-hydroxylase promoter. We conclude that physiological concentrations of insulin suppress bile acid synthesis by downregulation of cholesterol 7α-hydroxylase and sterol 27-hydroxylase gene transcription, and that this effect is mediated through a direct action of the hormone on the hepatocyte. These results may provide an explanation for the increased bile acid pool and excretion as found in humans with untreated diabetes mellitus and in experimental animals with insulin deficiency. Chemicals/CAS: cholesterol 7alpha monooxygenase, 9037-53-0; insulin, 9004-10-8; oxygenase, 9037-29-0, 9046-59-7; sterol 27 hydroxylase, 134712-57-5; Bile Acids and Salts; Chloramphenicol O-Acetyltransferase, EC 2.3.1.28; Cholesterol 7-alpha-Hydroxylase, EC 1.14.13.17; Cytochrome P-450 Enzyme System, 9035-51-2; cytochrome P-450C27/25, EC 1.14.-; Insulin, 11061-68-0; Membrane Glycoproteins; nuclear pore glycoprotein gp210; Nuclear Proteins; RNA, Messenger; Steroid Hydroxylases, EC 1.14.-

We showed previously that retinoids stimulate apolipoprotein A-I (apoA- I) synthesis in cultured cynomolgus hepatocytes only after a 24-h lag phase. Here we report on the biochemical background of the slow response, the requirement for high retinoic acid concentrations, and the involvement of different retinoid receptors. The time course of the effect of 10 μM all- trans retinoic acid (at-PA) on apoA-I mRNA levels and protein secretion were comparable, i.e., minor increases were observed after a 24-h incubation and mRNA levels were increased 2.2- and 3.5-fold after 48 h and 72 h, respectively. In contrast, apoA-I gene transcription was already increased (2.6-fold) after a 4-h incubation with 10 μM at-RA. At-RA disappeared rapidly from the cultures: after 2 h of incubation 40% of the added amount was left and after 24 h only 2%. RARβ mRNA and gene expression were increased after incubation with 10 nm at-RA, whereas RARer and RXRα mRNA levels and expression remained unchanged. No transcriptional activity and mRNA for other retinoid receptors were detectable. Both PAR-selective (TTNPB) and RXR-selective (3-methyl-TTNPB) agonists induced apoA-I synthesis at 1 and 10 μM. These results show that i) the slow increase in apoA-I secretion is caused by a slow increase of its mRNA level; ii) the apoA-I gene transcription in cynomolgus hepatocytes is induced rapidly by retinoids; iii) the added at-RA disappeared rapidly from the cultures, explaining the necessity for high initial concentrations; iv) RARα and/or RARβ and RXRα are involved in the activation of apoA-I expression by retinoids. Chemicals/CAS: 4-(2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid, 71441-28-6; alitretinoin, 5300-03-8; Apolipoprotein A-I; Benzoates; bexarotene; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; RNA, Messenger; Tetrahydronaphthalenes; Transcription Factors; Tretinoin, 302-79-4

The genome of the filamentous fungus Aspergillus niger is rich in genes encoding pectinases, a broad class of enzymes that have been extensively studied due to their use in industrial applications. The sequencing of the A. niger genome provided more knowledge concerning the individual pectinolytic genes, but little is known about the regulatory genes involved in pectin degradation. Understanding regulation of the pectinolytic genes provides a tool to optimize the production of pectinases in this industrially important fungus. This study describes the identification and characterization of one of the activators of pectinase-encoding genes, RhaR. Inactivation of the gene encoding this regulator resulted in down-regulation of genes involved in the release of L-rhamnose from the pectin substructure rhamnogalacturonan I, as well as catabolism of this monosaccharide. The rhaR disruptant was unable to grow on L-rhamnose, but only a small reduction in growth on pectin was observed. This is likely caused by the presence of a second, so far unknown regulator that responds to the presence of D-galacturonic acid. © 2014 Springer-Verlag. Chemicals/CAS: galacturonic acid, 14982-50-4, 685-73-4; pectin, 9000-69-5; pectin lyase, 9033-35-6; polygalacturonase, 9023-92-1, 9032-75-1; rhamnose, 10485-94-6, 3615-41-6

The gut microbiota is essential for numerous aspects of human health. However, the underlying mechanisms of many host-microbiota interactions remain unclear. The aim of this study was to characterize effects of the microbiota on host epithelium using a novel ex vivo model based on mouse ileal organoids. We have explored the transcriptional response of organoids upon exposure to short-chain fatty acids (SCFAs) and products generated by two abundant microbiota constituents, Akkermansia muciniphila and Faecalibacterium prausnitzii. We observed that A. muciniphila metabolites affect various transcription factors and genes involved in cellular lipid metabolism and growth, supporting previous in vivo findings. Contrastingly, F. prausnitzii products exerted only weak effects on host transcription. Additionally, A. muciniphila and its metabolite propionate modulated expression of Fiaf, Gpr43, histone deacetylases (HDACs), and peroxisome proliferator-activated receptor gamma (Pparγ), important regulators of transcription factor regulation, cell cycle control, lipolysis, and satiety. This work illustrates that specific bacteria and their metabolites differentially modulate epithelial transcription in mouse organoids. We demonstrate that intestinal organoids provide a novel and powerful ex vivo model for host-microbiome interaction studies. Importance We investigated the influence of the gut microbiota and microbially produced short-chain fatty acids (SCFAs) on gut functioning. Many commensal bacteria in the gut produce SCFAs, particularly butyrate, acetate, and propionate, which have been demonstrated to reduce the risk of gastrointestinal disorders. Organoids—small crypt-villus structures grown from ileal intestinal stem cells—were exposed to SCFAs and two specific gut bacteria. Akkermansia muciniphila, found in the intestinal mucus, was recently shown to have a favorable effect on the disrupted metabolism associated with obesity. Faecalibacterium prausnitzii is a commensal gut bacterium, the absence of which may be associated with Crohn’s disease. We showed that in our model, A. muciniphila induces stronger effects on the host than F. prausnitzii. We observed that A. muciniphila and propionate affect the expression of genes involved in host lipid metabolism and epigenetic activation or silencing of gene expression. We demonstrated that organoids provide a powerful tool for host-microbe interaction studies.

Pseudomonas putida KT2440-JD1 was derived from P. putida KT2440 after N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-mutagenesis and exposure to 3-fluorobenzoate (3-FB). The mutant was no longer able to grow using benzoate as a sole carbon source, but co-metabolized benzoate to cis, cis-muconate during growth on glucose, which accumulated in the growth medium. The specific production rate (qpm) was 0.18±0.03g cis, cis-muconate/(gDCWh) in continuous cultures, and increased to 1.4g cis, cis-muconate/(gDCWh) during wash-out cultivation. Transcriptome analysis showed that the cat operon was not induced in P. putida KT2440-JD1 in the presence of 5mM benzoate, due to a point mutation in the highly conserved DNA binding domain of the transcriptional regulator (catR) of the cat operon. The ben operon was highly expressed in the presence of benzoate in the mutant and its parental strain. This operon contains PP_3166 (catA2), which was shown to be a second catechol 1,2-dioxygenase besides catA. P. putida KT2440-JD1 is the first cis, cis-muconate-accumulating mutant that was characterized at the genetic level. The specific production rate achieved is at least eight times higher than those reported for other cis, cis-muconate-producing strains. © 2011 Elsevier B.V.

Chemical carcinogens may cause a multitude of effects inside cells, thereby affecting transcript levels of genes by direct activation of transcription factors (TF) or indirectly through the formation of DNA damage. As the temporal profiles of these responses may be profoundly different, examining time-dependent changes may provide new insights in TF networks related to cellular responses to chemical carcinogens. Therefore, we investigated in human hepatoma cells gene expression changes caused by benzo[a]pyrene at 12 time points after exposure, in relation to DNA adduct and cell cycle. Temporal profiles for functional gene sets demonstrate both early and late effects in up- and downregulation of relevant gene sets involved in cell cycle, apoptosis, DNA repair, and metabolism of amino acids and lipids. Many significant transcription regulation networks appeared to be around TF that are proto-oncogenes or tumor suppressor genes. The time series analysis tool Short Time-series Expression Miner (STEM) was used to identify time-dependent correlation of pathways, gene sets, TF networks, and biological parameters. Most correlations are with DNA adduct levels, which is an early response, and less with the later responses on G1 and S phase cells. The majority of the modulated genes in the Reactome pathways can be regulated by several of these TF, e.g., 73% by nuclear factor-kappa B and 34-42% by c-MYC, SRF, AP1, and E2F1. All these TF can also regulate one or more of the others. Our data indicate that a complex network of a few TF is responsible for the majority of the transcriptional changes induced by BaP. This network hardly changes over time, despite that the transcriptional profiles clearly alter, suggesting that also other regulatory mechanisms are involved. © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.

Solid-state fermentation (SSF) with Aspergillus oryzae results in high levels of secreted protein. However, control mechanisms of gene expression in SSF have been only poorly studied. In this study we show that both glucoamylase (glaB) and protease (alpA, nptB) genes are highly expressed during surface cultivation on wheat-based solid medium, and even higher during cultivation on wheat kernels. In wheat-based liquid medium, low levels of gene expression are observed. Typical SSF cultivation conditions, such as low water activity and the formation of aerial hyphae, did not contribute to the high-level gene expression on wheat-based solid medium. Analysis of wheat-based solid and liquid cultivations showed differences in carbon and nitrogen utilisation and external pH. The results presented show that the difference in regulation of transcription of the alpA and nptB genes in wheat-based liquid and solid medium could be pH dependent, involving a pH-dependent transcription regulator. The results obtained suggest that the difference in regulation of transcription of the glaB gene in wheat-based liquid and solid medium is caused by a difference in carbohydrate degradation and consumption under the different culture conditions. © Springer-Verlag 2004.

We describe a new endoplasmic reticulum (ER)-associated stress response in the filamentous fungus Aspergillus niger. The inhibition of protein folding within the ER leads to cellular responses known collectively as the unfolded protein response (UPR) and we show that the selective transcriptional downregulation of the gene encoding glucoamylase, a major secreted protein, but not two non-secreted proteins, is an additional consequence of ER stress. The transcriptional downregulation effect is shown by nuclear run-on studies to be at the level of transcription, rather than mRNA stability, and is found to be mediated through the promoter of gIaA in a region more than 1 kb upstream of the translational start. The inhibition of protein folding in the ER can be induced in a variety of ways. We examined the effects of dithiothreltol (DTT), a reducing agent that causes the formation of unfolded proteins. Although a general downregulation of transcription was seen with DTT treatment, we show that selective downregulation was observed with the gIaA, gene compared with genes encoding the non-secreted proteins γ-actin and glyceraldehyde 3′-phosphate dehydrogenase. The DTT-treated fungal cells also showed evidence for the induction of the UPR because expression of bipA and pdiA, encoding an ER-resident chaperone and foldase, respectively, are upregulated and splicing of hscA, the gene encoding the transcription factor responsible for induction of the UPR, occurs allowing the production of an active HacA protein. As a preliminary attempt to investigate if the transcriptional downregulation effect was mediated through HacA (i.e. part of the UPR), we examined ER stress induced through antisense technology to lower the level of PDI in the ER of A. niger. Although the transcription of gIaA was attenuated in that strain of A. niger, UPR was not evident, suggesting that the transcriptional downregulation mechanism is controlled differently from the UPR.

Rats were exposed to three levels of bromobenzene, sampled at 6, 24, and 48 h, and liver gene expression profiles were determined to identify dose and time-related changes. Expression of many genes changed transiently, and dependent on the dose. Few changes were identified after 6 h, but many genes were differentially expressed after 24 h, while after 48 h, only the high dose elicited large effects. Differentially expressed genes were involved in drug metabolism (upregulated GSTs, mEH, NQO1, Mrps, downregulated CYPs, sulfotransferases), oxidative stress (induced HO-1, peroxiredoxin, ferritin), GSH depletion (induced GCS-1, GSTA, GSTM) the acute phase response, and in processes like cholesterol, fatty acid and protein metabolism, and intracellular signaling. Trancriptional regulation via the electrophile and sterol response elements seemed to mediate part of the response to bromobenzene. Recovery of the liver was suggested in response to BB by the altered expression of genes involved in protein synthesis and cytoskeleton rearrangement. Furthermore, after 48 h, rats in the mid dose group showed no toxicity, and gene expression patterns resembled the normal situation. For certain genes (e.g., CYP4A, metallothioneins), intraday variation in expression levels was found, regardless of the treatment. Selected cDNA microarray measurements were confirmed using the specific and sensitive branched DNA signal amplification assay. © Society of Toxicology 2004; all rights reserved.

The interaction between the human body and nutrition is an extremely complex process involving multi-organ physiology with molecular mechanisms on all levels of regulation (genes, gene expression, proteins, metabolites). Only with the recent technology push have nutritional scientists been able to address this complexity. Both the challenges and promises that are offered by the merge of 'biomics' technologies and mechanistic nutrition research are huge, but will eventually evolve in a new nutrition research concept: nutritional systems biology. This review describes the principles and technologies involved in this merge. Using nutrition research examples, including gene expression modulation by carbohydrates and fatty acids, this review discusses applications as well as limitations of genomics, transcriptomics, proteomics, metabolomics, and systems biology. Furthermore, reference is made to gene polymorphisms that underlie individual differences in nutrient utilization, resulting in, e.g., different susceptibility to develop obesity. Copyright © 2005 S. Karger AG, Basel/ILSI Europe.

In previous work we have demonstrated suppression of cholesterol 7α-hydroxylase by bile acids at the level of mRNA and transcription, resulting in a similar decline in bile acid synthesis in cultured rat hepatocytes. In view of the substantial contribution of the 'alternative' or '27-hydroxylase' route to total bile acid synthesis, as demonstrated in cultured rat hepatocytes and in vivo in humans, we here evaluate the effects of various bile acids commonly found in bile of rats on the regulation of sterol 27-hydroxylase in cultured rat hepatocytes. Addition of taurocholic acid, the predominant bile acid in rat bile, to the culture medium of rat hepatocytes resulted in a 72% inhibition of sterol 27-hydroxylase activity. The effect was exerted at the level of sterol 27-hydroxylase mRNA, showing a time- and dose-dependent decline with a maximal suppression (-75%) at 50 μM taurocholic acid after 24 h of culture. The decline in mRNA followed first-order kinetics with an apparent half-life of 13 h. Under these conditions cholesterol 7α-hydroxylase mRNA (-91%) and bile acid synthesis (i.e. chenodeoxycholic and β-muricholic acid, -81%) were also maximally suppressed. In contrast, no change was found in the level of lithocholic acid 6β-hydroxylase mRNA. Assessment of the transcriptional activity of a number of genes involved in routing of cholesterol towards bile acids showed similar suppressive effects of taurocholate on expression of the sterol 27-hydroxylase and cholesterol 7α-hydroxylase genes (-43% and -42% respectively), whereas expression of the lithocholic 6β-hydroxylase gene was not affected. Taurocholic acid and unconjugated cholic acid were equally as effective in suppressing sterol 27-hydroxylase mRNA. The more hydrophobic bile acids, chenodeoxycholic acid and deoxycholic acid also produced a strong inhibition of 57% and 76% respectively whereas the hydrophilic β-muricholic acid was not active. We conclude that (1) a number of bile acids, at physiological concentrations, suppress sterol 27-hydroxylase by down-regulation of sterol 27-hydroxylase mRNA and transcriptional activity and (2) co-ordinated suppression of both sterol 27-hydroxylase and cholesterol 7α-hydroxylase results in inhibition of bile acid synthesis in cultured rat hepatocytes. Chemicals/CAS: chenodeoxycholic acid, 474-25-9; cholesterol 7alpha monooxygenase, 9037-53-0; cholic acid, 32500-01-9, 361-09-1, 81-25-4; deoxycholic acid, 83-44-3; lithocholic acid, 434-13-9; oxygenase, 9037-29-0, 9046-59-7; taurocholic acid, 145-42-6, 59005-70-8, 81-24-3; Adenosine Triphosphate, 56-65-5; Bile Acids and Salts; Cholesterol 7-alpha-Hydroxylase, EC 1.14.13.17; Cytochrome P-450 Enzyme System, 9035-51-2; cytochrome P-450C27/25, EC 1.14.-; Oxidoreductases, EC 1.; RNA, Messenger; Steroid Hydroxylases, EC 1.14.-; Taurocholic Acid, 81-24-3

We have used primary monolayer cultures of rat hepatocytes to study the effects of physiological concentrations of various bile acids, commonly found in bile of normal rats, on the mechanism of regulation of cholesterol 7α-hydroxylase and bile acid synthesis. Addition of taurocholic acid, the most predominant bile acid in rat bile, to the culture medium suppressed cholesterol 7α-hydroxylase activity and mRNA time- and dose-dependently. The decrease in enzyme activity paralleled the changes in mRNA. Maximal suppression of cholesterol 7α-hydroxylase mRNA (-91%) and enzyme activity (-89%) was observed after a 16 h incubation period with 50 uM taurocholic acid. The declines in mRNA and enzyme caused by taurocholic acid were tightly coupled and followed first-order kinetics with a half-life of 4 h. Transcriptional activity, as assessed with nuclear run-on assays, was decreased by 44% at 50 μM taurocholic acid. Mass production of bile acids (chenodeoxycholic acid and β-muricholic acid) was inhibited to a similar extent as the cholesterol 7α-hydroxylase when different concentrations of taurocholic acid were used, giving maximal inhibition (-81%) at 50 μM taurocholic acid. Glycocholic acid and unconjugated cholic acid were equally effective as taurocholic acid in suppressing cholesterol 7α-hydroxylase mRNA. The more hydrophobic bile acids (chenodeoxycholic acid and deoxycholic acid) showed profound suppression of the cholesterol 7α-hydroxylase mRNA by 85% and 75% respectively, whereas the other trihydroxy bile acids in rat bile, α- and β-muricholic acid, were not or only marginally active. We conclude that rat bile acids, in particular the more hydrophobic ones, in concentrations commonly observed in portal blood, exert negative feedback control at the level of cholesterol 7α-hydroxylase mRNA in cultured rat hepatocytes through a direct effect on the hepatocytes, and that down-regulation of transcription is only one of the mechanisms involved in this regulation. Chemicals/CAS: chenodeoxycholic acid, 474-25-9; cholesterol 7alpha monooxygenase, 9037-53-0; cholic acid, 32500-01-9, 361-09-1, 81-25-4; deoxycholic acid, 83-44-3; glycocholic acid, 475-31-0; taurocholic acid, 145-42-6, 59005-70-8, 81-24-3; Bile Acids and Salts; Chenodeoxycholic Acid, 474-25-9; Cholesterol 7-alpha-Hydroxylase, EC 1.14.13.17; Cholic Acid, 81-25-4; Cholic Acids; Glycocholic Acid, 475-31-0; muricholic acid, 39016-49-4; RNA, Messenger; Taurocholic Acid, 81-24-3

As a soil fungus, Aspergillus niger can metabolize a wide variety of carbon sources, employing sets of enzymes able to degrade plant-derived polysaccharides. In this study the genome sequence of A. niger strain CBS 513.88 was surveyed, to analyse the gene/enzyme network involved in utilization of the plant storage polymer inulin, and of sucrose, the substrate for inulin synthesis in plants. In addition to three known activities, encoded by the genes suc 1 (invertase activity; designated sucA), inuE (exo-inulinase activity) and inuAlinuB (endo-inulinase activity), two new putative invertase-like proteins were identified. These two putative proteins lack N-terminal signal sequences and therefore are expected to be intracellular enzymes. One of these two genes, designated sucB, is expressed at a low level, and its expression is up-regulated when A. niger is grown on sucrose- or inulin-containing media. Transcriptional analysis of the genes encoding the sucrose- (sucA) and inulin-hydrolysing enzymes (inuA and inuE) indicated that they are similarly regulated and all strongly induced on sucrose and inulin. Analysis of a ΔcreA mutant strain of A. niger revealed that expression of the extracellular inulinolytic enzymes is under control of the catabolite repressor CreA. Expression of the inulinolytic enzymes was not induced by fructose, not even in the ΔcreA background, indicating that fructose did not act as an inducer. Evidence is provided that sucrose, or a sucrose-derived intermediate, but not fructose, acts as an inducer for the expression of inulinolytic genes in A. niger. © 2006 SGM. Chemicals / CAS: glycosidase, 9032-92-2; inulin, 9005-80-5; sucrose, 122880-25-5, 57-50-1; DNA, Fungal; Fructose, 30237-26-4; Inulin, 9005-80-5; Protein Sorting Signals; RNA, Fungal; RNA, Messenger; Sucrose, 57-50-1; beta-Fructofuranosidase, 3.2.1.26. Molecular Sequence Numbers: GENBANK: BAA12321, BAA25684, BAA33797, BAA33798, BAB19132, BAB67771, BAC16218, BAD01476, CAA04131, CAA07345, CAA40488, CAA56684, CAA73208, CAB89083, CAC28747, CAC44220, EAA59102, EAA61090, EAA69589, EAA69879, EAA71892, EAA72735, EAA78236, EAK82399, EAK84508, EAL85540, EAL86248, EAL86253, EAL87222, P00724, S33920, XP_360436, XP_360455, XP_365805, XP_367933, XP_369679, DQ233218, DQ233219, DQ233220, DQ233221, DQ233222, DQ233223;

The filamentous ascomycete Aspergillus niger is well known for its ability to produce a large variety of enzymes for the degradation of plant polysaccharide material. A major carbon and energy source for this soil fungus is starch, which can be degraded by the concerted action of α-amylase, glucoamylase and α-glucosidase enzymes, members of the glycoside hydrolase (GH) families 13, 15 and 31, respectively. In this study we have combined analysis of the genome sequence of A. niger CBS 513.88 with microarray experiments to identify novel enzymes from these families and to predict their physiological functions. We have identified 17 previously unknown family GH13, 15 and 31 enzymes in the A. niger genome, all of which have orthologues in other aspergilli. Only two of the newly identified enzymes, a putative α-glucosidase (AgdB) and an α-amylase (AmyC), were predicted to play a role in starch degradation. The expression of the majority of the genes identified was not induced by maltose as carbon source, and not dependent on the presence of AmyR, the transcriptional regulator for starch degrading enzymes. The possible physiological functions of the other predicted family GH13, GH15 and GH31 enzymes, including intracellular enzymes and cell wall associated proteins, in alternative α-glucan modifying processes are discussed. © 2008 The Author(s).

Background: In bacteriology, the ability to grow in selective media and to form colonies on nutrient agar plates is routinely used as a retrospective criterion for the detection of living bacteria. However, the utilization of indicators for bacterial viability-such as the presence of specific transcripts or membrane integrity-would overcome bias introduced by cultivation and reduces the time span of analysis from initiation to read out. Therefore, we investigated the correlation between transcriptional activity, membrane integrity and cultivation-based viability in the Gram-positive model bacterium Bacillus subtilis. Results: We present microbiological, cytological and molecular analyses of the physiological response to lethal heat stress under accurately defined conditions through systematic sampling of bacteria from a single culture exposed to gradually increasing temperatures. We identified a coherent transcriptional program including known heat shock responses as well as the rapid expression of a small number of sporulation and competence genes, the latter only known to be active in the stationary growth phase. Conclusion: The observed coordinated gene expression continued even after cell death, in other words after all bacteria permanently lost their ability to reproduce. Transcription of a very limited number of genes correlated with cell viability under the applied killing regime. The transcripts of the expressed genes in living bacteria - but silent in dead bacteria-include those of essential genes encoding chaperones of the protein folding machinery and can serve as molecular biomarkers for bacterial cell viability. © 2008 Kort et al; licensee BioMed Central Ltd. Chemicals / CAS: Biological Markers; RNA, Bacterial