[Abdominal pain and ascites formation in a 72-year-old woman]. / Abdominale Schmerzen und neu aufgetretener Aszites bei einer 72-jährigen Patientin.

A 72-year-old woman presented with abdominal pain after micturition. Abdominal ultrasound screening revealed ascites associated with acute renal failure. Paracentesis of the peritoneal fluid was performed. Biochemical analysis indicated a peritoneal transsudate and increased creatinine. Cystoscopy detected a rupture of the urinary bladder. Catheterization and antibiotic therapy resulted in an improvement of pain and closure of the hole in the urinary bladder wall. Several different disorders can induce a rupture of the urinary bladder. In this case, severe chronic constipation was the most probable causative disease.

Nitrate and nitrite control of respiratory nitrate reduction in denitrifying Pseudomonas stutzeri by a two-component regulatory system homologous to NarXL of Escherichia coli.

Bacterial denitrification is expressed in response to the concurrent exogenous signals of low-oxygen tension and nitrate or one of its reduction products. The mechanism by which nitrate-dependent gene activation is effected was investigated in the denitrifying bacterium Pseudomonas stutzeri ATCC 14405. We have identified and isolated from this organism the chromosomal region encoding the two-component sensor-regulator pair NarXL and found that it is linked with the narG operon for respiratory nitrate reductase. The same region encodes two putative nitrate or nitrite translocases, NarK and NarC (the latter shows the highest similarity to yeast [Pichia] and plant [Nicotiana] nitrate transporters), and the nitrate-regulated transcription factor, DnrE, of the FNR family. The roles of NarX and NarL in nitrate respiration were studied with deletion mutants. NarL activated the transcription of narG, narK, and dnrE but did not affect the denitrification regulons for the respiratory substrates nitrite, nitric oxide, and nitrous oxide. The promoters of narG, narK, and dnrE carry sequence motifs, TACYYMT, which correspond to the NarL recognition sequence established for Escherichia coli. The cellular response toward nitrate and nitrite was mediated by the sensor protein NarX, which discriminated weakly between these oxyanions. Our data show that the NarXL two-component regulatory system has been incorporated into the bacterial denitrification process of P. stutzeri for selective regulation of nitrate respiration.

Multiple transcription factors of the FNR family in denitrifying Pseudomonas stutzeri: characterization of four fnr-like genes, regulatory responses and cognate metabolic processes.

Pseudomonas stutzeri is a facultative anaerobic bacterium with the capability of denitrification. In searching for regulators that control the expression of this trait in response to oxygen withdrawal, we have found an unprecedented multiplicity of four genes encoding transcription factors of the FNR family. The fnrA gene encodes a genuine FNR-type regulator, which is expressed constitutively and controls the cytochrome cbb3-type terminal oxidase (the cco operon), cytochrome c peroxidase (the ccp gene) and the oxygen-independent coproporphyrinogen III oxidase (the hemN gene), in addition to its previously demonstrated role in arginine catabolism (the arc operon). The fnr homologues dnrD, dnrE and dnrS encode regulators of a new subgroup within the FNR family. Their main distinctive feature is the lack of cysteine residues for complexing the [4Fe-4S] centre of redox-active FNR-type regulators. However, they form a phylogenetic lineage separate from the FixK branch of FNR proteins, which also lack this cysteine signature. We have studied the expression of the dnr genes under aerobic, oxygen-limited and denitrifying conditions. DnrD is a key regulator of denitrification by selective activation of the genes for cytochrome cd1 nitrite reductase and NO reductase. The dnrD gene is part of the 30 kb region carrying denitrification genes of P. stutzeri. Transcription of dnrD was activated in O2-limited cells and particularly strongly in denitrifying cells, but was not under the control of FnrA. In response to denitrifying growth conditions, dnrD was transcribed as part of an operon together with genes downstream and upstream of dnrD. dnrS was found about 9 kb upstream of dnrD, next to the nrdD gene for anaerobic ribonucleotide reductase. The transcription of dnrS required FnrA in O2-limited cells. Mutation of dnrS affected nrdD and the expression of ferredoxin I as an element of the oxidative stress response. The dnrE gene is part of the nar region encoding functions for respiratory nitrate reduction. We found the highest amount of dnrE transcripts in aerobically nitrate-challenged cells. The gene was transcribed from two promoters, P1 and P2, of which promoter P1 was under the control of the nitrate response regulator NarL. The multiplicity of FNR factors in P. stutzeri underlines the versatility of the FNR scaffold to serve for transcriptional regulation directed at anaerobic or nitrate-activated metabolic processes.

Kinetics of nirS expression (cytochrome cd1 nitrite reductase) in Pseudomonas stutzeri during the transition from aerobic respiration to denitrification: evidence for a denitrification-specific nitrate- and nitrite-responsive regulatory system.

After shifting an oxygen-respiring culture of Pseudomonas stutzeri to nitrate or nitrite respiration, we directly monitored the expression of the nirS gene by mRNA analysis. nirS encodes the 62-kDa subunit of the homodimeric cytochrome cd1 nitrite reductase involved in denitrification. Information was sought about the requirements for gene activation, potential regulators of such activation, and signal transduction pathways triggered by the alternative respiratory substrates. We found that nirS, together with nirT and nirB (which encode tetra- and diheme cytochromes, respectively), is part of a 3.4-kb operon. In addition, we found a 2-kb monocistronic transcript. The half-life of each of these messages was approximately 13 min in denitrifying cells with a doubling time of around 2.5 h. When the culture was subjected to a low oxygen tension, we observed a transient expression of nirS lasting for about 30 min. The continued transcription of the nirS operon required the presence of nitrate or nitrite. This anaerobically manifested N-oxide response was maintained in nitrate sensor (NarX) and response regulator (NarL) knockout strains. Similar mRNA stability and transition kinetics were observed for the norCB operon, encoding the NO reductase complex, and the nosZ gene, encoding nitrous oxide reductase. Our results suggest that a nitrate- and nitrite-responsive regulatory circuit independent of NarXL is necessary for the activation of denitrification genes.

The requirement of RpoN (sigma factor sigma54) in denitrification by Pseudomonas stutzeri is indirect and restricted to the reduction of nitrite and nitric oxide.

The rpoN region of Pseudomonas stutzeri was cloned, and an rpoN null mutant was constructed. RpoN was not essential for denitrification in this bacterium but affected the expression levels and enzymatic activities of cytochrome cd1 nitrite reductase and nitric oxide reductase, whereas those of respiratory nitrate reductase and nitrous oxide reductase were comparable to wild-type levels. Since the transcription of the structural genes nirS and norCB, coding for nitrite reductase and the nitric oxide reductase complex, respectively, proceeded unabated, our data indicate a posttranslational process for the two key enzymes of denitrification depending on RpoN.

In vivo binding of proteins to stably integrated MMTV DNA in murine cell lines: occupancy of NFI and OTF1 binding sites in the absence and presence of glucocorticoids.

Activation of expression at the mouse mammary tumor virus (MMTV) promoter is thought to be controlled by nucleosome positioning. On stably integrated MMTV DNA, the long terminal repeat (LTR) region is organized in a phased array of nucleosomes which allegedly occludes transcription factors such as NFI from binding. NFI only binds to the promoter region when the ordered nucleosome structure is apparently disrupted by activated steroid hormone receptors in hormone induced transcription. In certain cell lines, binding sites for the transcription factors NFI and OTF1 are however required for hormone-independent expression of MMTV. We have used stably transfected mouse NIH3T3 and GR cells that exhibit detectable MMTV expression in the absence of hormone for in vivo determination of proteins binding to the MMTV promoter. Here, we present in vivo dimethyl sulfate footprinting data that show that the NFI and OTF binding sites are permanently occupied in vivo in these cells. The contacting guanine residues identified in vivo were demonstrated in in vitro methylation interference assays to correspond to binding by NFI and OTF1. These results demonstrate a novel feature of transcription factor occupancy at the MMTV LTR promoter.

Regulation of expression of mouse mammary tumor virus through sequences located in the hormone response element: involvement of cell-cell contact and a negative regulatory factor.

Mouse mammary tumor virus (MMTV) is a latently oncogenic retrovirus responsible for the neoplastic transformation of mammary epithelial cells. Its expression is regulated by steroids, polypeptide growth factors, and cell-type-specific factors. Using GR mouse mammary cells and NIH 3T3 fibroblasts stably transfected with chimeric constructs of the long terminal repeat region of MMTV, we have demonstrated a novel mechanism of cell-type-specific expression of this virus. In confluent monolayer cultures that permit cell-cell interaction, MMTV long terminal repeat expression is positively regulated by sequences within the hormone response element (HRE) that bind the transcription factors CTF/NFI and OTFI. Although these factors are present in NIH 3T3 cells, MMTV expression in these cells is not regulated by cell density. This is partially due to a negative regulatory factor that binds sequences between -164 and -151 in the HRE. Mutations that destroy the binding site for this factor restored in part the cell density-regulated expression of MMTV to NIH 3T3 fibroblasts. The HRE is thus a central coordinator of regulatory pathways that positively or negatively influence the expression of MMTV.

A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.

Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites.

A new cAMP response element in the transcribed region of the human c-fos gene.

In NIH 3T3 cells the c-fos gene is induced rapidly and transiently by cAMP. As shown by the analysis of 3T3 cells stably transfected with promoter mutants of the human c-fos gene this induction does not depend on the dyad symmetry element (position -320 to -300), but involves at least two other non-related sites: an element located around position -60 resembling the cAMP response element of the fibronectin and somatostatin genes (which has been described before), and an element located between positions +18 and +38. Destruction of one or the other element in the c-fos gene reduces cAMP inducibility. The cAMP response of c-fos promoter CAT gene constructs also depends on these elements in transient transfection assays. When cloned in front of the albumin TATA box, both elements independently mediate cAMP inducibility. These elements do not bind the same protein as shown in gel retardation analyses, suggesting that two different cAMP inducible factors mediate the activation of the c-fos gene by cAMP.