Volume 386, Issue 6

MDR1/P-glycoprotein is an efflux transporter determining the absorption and presystemic elimination of many xenobiotics in the gut. Thus, interindividual differences in MDR1 expression may affect the efficacy of drug treatment. The expression of MDR1 is partially controlled by the pregnane X receptor (PXR), which mediates induction by many xenobiotics. Since it has been described that the nuclear receptors PXR and constitutive androstane receptor (CAR) can bind to the same binding sites, we investigated the role of CAR in the regulation of MDR1 gene expression. We demonstrate here by gel shift and transfection experiments that CAR binds to distinct nuclear receptor response elements in the -7.8 kbp enhancer of MDR1 and transactivates MDR1 expression through DR4 motifs to which the receptor binds as a heterodimer with RXR or as a monomer, respectively. Expression of the endogenous MDR1 gene is elevated in cells stably expressing CAR, thus arguing for the functional relevance of CAR-dependent activation of MDR1 . The physiological relevance of the regulation of MDR1 by CAR is further suggested by correlation of the expression of CAR and MDR1 in the human small intestine. In summary, our data suggest that CAR plays a role in the regulation of intestinal MDR1 expression.

Kinins are important mediators in cardiovascular homeostasis, inflammation, and nociception. Two kinin receptors have been described, B 1 and B 2 . The B 1 receptor is normally absent in healthy tissues, but is highly induced under pathological conditions. To understand the molecular mechanism of B 1 receptor up-regulation, we determined the mouse B 1 receptor gene structure, isolated and characterized the promoter region and studied its transcriptional regulation. The mouse B 1 receptor gene contains two exons (with the entire coding region located in the second exon) and a TATA-less promoter with multiple transcription start sites. A 7.7-kbp portion of the 5′-flanking region was examined for promoter activity in vascular smooth muscle cells (VSMCs). A minimal 92-bp fragment, located immediately upstream of the transcription start region, exerted basal and lipopolysaccharide (LPS)-inducible transcription activity in the sense and antisense orientation, and was thereby identified as an enhancer element. Nuclear extracts from VSMCs showed basal and LPS-inducible binding activity of nuclear factor (NF)-κB at this sequence. B 1 receptor transcription activation in response to LPS was abolished by cotransfection with IκBαΔN, an NF-κB repressor. In summary, our results reveal the structure of the mouse B 1 receptor gene and the involvement of NF-κB in the inducible mouse kinin B 1 receptor expression under pathological conditions.

Ribosomal RNAs in E. coli are transcribed from seven operons, which are highly conserved in their organization and sequence. However, the upstream regulatory DNA regions differ considerably, suggesting differences in regulation. We have therefore analyzed the conformation of all seven DNA elements located upstream of the major E. coli rRNA P1 promoters. As judged by temperature-dependent gel electrophoresis with isolated DNA fragments comprising the individual P1 promoters and the complete upstream regulatory regions, all seven rRNA upstream sequences are intrinsically curved. The degree of intrinsic curvature was highest for the rrnB and rrnD fragments and less pronounced for the rrnA and rrnE operons. Comparison of the experimentally determined differences in curvature with programs for the prediction of DNA conformation revealed a generally high degree of conformity. Moreover, the analysis showed that the center of curvature is located at about the same position in all fragments. The different upstream regions were analyzed for their capacity to bind the transcription factors FIS and H-NS, which are known as antagonists in the regulation of rRNA synthesis. Gel retardation experiments revealed that both proteins interact with the upstream promoter regions of all seven rDNA fragments, with the affinities of the different DNA fragments for FIS and H-NS and the structure of the resulting complexes deviating considerably. FIS binding was non-cooperative, and at comparable protein concentrations the occupancy of the different DNA fragments varied between two and four binding sites. In contrast, H-NS was shown to bind cooperatively and intermediate states of occupancy could not be resolved for each fragment. The different gel electrophoretic mobilities of the individual DNA/protein complexes indicate variable structures and topologies of the upstream activating sequence regulatory complexes. Our results are highly suggestive of differential regulation of the individual rRNA operons.

The enzyme L-asparaginase is a crucial component in the treatment of acute lymphoblastic leukemia (ALL). As all asparaginases in clinical use are derived from microorganisms, immunological reactions are the most important adverse events associated with asparaginase treatment. Two different methods, phage display and the SPOTs method, were used for the determination of clinically relevant epitopes. Comparison of the results showed that essentially the same domains were identified by the two methods, and thus ascertainment of relevant epitopes can be assumed. Determination of the specificity of the epitopes will be performed with serum from patients with different modes of immunological reactions and from individuals without evidence of an immune response after asparaginase administration.

The members of the H1 histone family can be classified into three groups, which are the main class subtypes expressed in somatic cells, the developmental- and tissue-specific subtypes, and the replacement subtype H1°. Until now, the subtype H1x was not classified, since it has not yet been thoroughly examined. The results of this study show that H1x shares similarities but also exhibits slight differences in its biochemical behaviour in comparison to the main class H1 histones. In HeLa cells it is located in the nucleus and partially associated with nucleosomes. Nevertheless, it is, like H1°, mainly located in chromatin regions that are not affected by micrococcal nuclease digestion. Further common features of H1x and the replacement histone H1° are that the genes of both subtypes are solitarily located and give rise to polyadenylated mRNA. However, comparison of the inducibility of their expression revealed that their genes are regulated differentially.

Cinnamoyl-CoA reductase (CCR) is responsible for the CoA ester→aldehyde conversion in monolignol biosynthesis, which can divert phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of lignin biosynthesis in wheat ( Triticum aestivum L.), a cDNA encoding CCR was isolated and named Ta-CCR2. DNA hybridization analyses demonstrated that the Ta-CCR2 gene exists in three copies in the wheat genome. RNA blot hybridization indicated that Ta-CCR2 was expressed most abundantly in root and stem tissues that were in the process of lignification. The secondary and three-dimensional structures of Ta-CCR2 were analyzed by molecular modeling. Recombinant Ta-CCR2 protein purified from E. coli converted feruloyl CoA, 5-OH-feruloyl CoA, sinapoyl CoA and caffeoyl CoA with almost similar efficiency, suggesting that it is involved in both G and S lignin synthesis. Ta-CCR2 had a very low V max value for 4-coumaroyl CoA, which may serve as a mechanism to control metabolic flux to H lignin in vivo . Furthermore, the reaction mechanism of Ta-CCR2 was analyzed in relation to its possible three-dimensional structure. The activity of Ta-CCR2 in relation to lignin biosynthesis is discussed.

Bauhinia bauhinoides cruzipain inhibitor (BbCI) and Bauhinia bauhinioides kallikrein inhibitor (BbKI) are cysteine and serine proteinase inhibitors structurally homologous to plant Kunitz-type inhibitors, but are devoid of disulfide bridges. Based on cDNA sequences, we found that BbKI and BbCI are initially synthesized as a prepropeptide comprising an N-terminal signal peptide (19 residues), the mature protein (164 residues) and a C-terminal targeting peptide (10 residues). Partial cDNAs encoding the mature enzymes plus N-terminal His-tags and thrombin cleavage sites were expressed in E. coli and the soluble proteins were purified by one-step nickel affinity chromatography. After thrombin cleavage, both proteins exhibited potent inhibitory activities toward their cognate proteinases like the wild-type proteins. BbCI inhibits human neutrophil elastase ( K i(app) 5.3 nM), porcine pancreatic elastase ( K i(app) 40 nM), cathepsin G ( K i(app) 160 nM) and the cysteine proteinases cruzipain ( K i(app) 1.2 nM), cruzain ( K i(app) 0.3 nM) and cathepsin L ( K i(app) 2.2 nM), while BbKI strongly inhibits plasma kallikrein ( K i(app) 2.4 nM) and plasmin ( K i(app) 33 nM). Circular dichroism spectra of BbCI and BbKI were in agreement with the β-trefoil fold described for Kunitz inhibitors. The inhibitory potency of both BbCI- and BbKI-type inhibitors suggests that other, non-covalent interactions may compensate for the lack of disulfide bridges.

The conversion of the α-helical, cellular isoform of the prion protein (PrP C ) to the insoluble, β-sheet-rich, infectious, disease-causing isoform (PrP Sc ) is the fundamental event in the prion diseases. The C-terminal fragment of PrP Sc (PrP 27–30) is formed by limited proteolysis and retains infectivity. Unlike full-length PrP Sc , PrP 27–30 polymerizes into rod-shaped structures with the ultra-structural and tinctorial properties of amyloid. To study the folding of PrP, both with respect to the formation of PrP Sc from PrP C and the assembly of rods from PrP 27–30, we solubilized Syrian hamster (sol SHa) PrP 27–30 in low concentrations (0.2%) of sodium dodecyl sulfate (SDS) under conditions previously used to study the structural transitions of this protein. Sol SHaPrP 27–30 adopted a β-sheet-rich structure at SDS concentrations between 0.02% and 0.04% and remained soluble. Here we report that NaCl stabilizes SHaPrP 27–30 in a soluble, β-sheet-rich state that allows fibril assembly to proceed over several weeks. Under these conditions, fibril formation occurred not only with sol PrP 27–30, but also with native SHaPrP C . Addition of sphingolipids seems to increase fibril growth. When recombinant (rec) SHaPrP(90–231) was exposed to low concentrations of SDS, similar to those used to polymerize sol SHaPrP 27–30 in the presence of 250 mM NaCl, fibril formation occurred regularly. When fibrils formed from PrP 27–30 or PrP C were bioassayed in transgenic mice overexpressing full-length SHaPrP, no infectivity was obtained, whereas amyloid fibrils formed of rec mouse PrP(89–230) were infectious. At present, it cannot be determined whether the lack of infectivity is caused by a difference in the structure of the fibrils or in the bioassay conditions.

We describe the isolation and characterisation of an insulinotropic peptide from the skin secretions of Agalychnis calcarifer frogs. Peptides in crude secretions obtained by mild electrical stimulation from the dorsal skin surface were purified by reversed-phase HPLC, yielding fractions in two zones with insulin-releasing activity ( p <0.001). The peaks showing greatest in vitro insulin-releasing activity were subsequently purified to homogeneity, revealing a novel insulinotropic 13-amino-acid (1653.2 Da) peptide with the primary structure RRKPLFPLIPRPK (RK-13). A database search for RK-13 showed 53.8% similarity with the N-terminal region of proline-arginine-rich antimicrobial peptide (PR-39). Synthetic RK-13 stimulated insulin release in a dose-dependent, glucose-sensitive manner, exerting its effects through a cyclic AMP-protein kinase A pathway independent of pertussis toxin-sensitive G proteins. Unlike PR-39, RK-13 lacks antimicrobial effects on the growth of yeast, and Gram-positive and Gram-negative bacteria. Our data indicate that skin secretions of Agalychnis calcarifer frogs contain insulin-releasing peptides, including RK-13, which merit further investigation as insulin secretagogues.

The first low-molecular-mass metalloprotease presenting prothrombin activating activity was purified from Bothrops insularis venom and named insularinase A. It is a single-chain protease with a molecular mass of 22 639 Da. cDNA sequence analysis revealed that the disintegrin domain of the precursor protein is post-translationally processed, producing the mature insularinase A. Analysis of its deduced amino acid sequence showed a high similarity with several fibrin(ogen)olytic metalloproteases and only a moderate similarity with prothrombin activators. However, SDS-PAGE of prothrombin after activation by insularinase A showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin independently of the prothrombinase complex. In addition, insularinase A activates factor X and hydrolyses fibrinogen and fibrin. Chelating agents fully inhibit all insularinase A activities. Insularinase A induced neither detachment nor apoptosis of human endothelial cells and was also not able to trigger an endothelial proinflammatory cell response. Nitric oxide and prostacyclin levels released by endothelial cells were significantly increased after treatment with insularinase A. Our results show that, although its primary structure is related to class P-I fibrin(ogen)olytic metalloproteases, insularinase A is functionally similar to group A prothrombin activators.

Polyphenol oxidase (PPO) was extracted from beet root, in both soluble and membrane fractions, and in both cases the enzyme was in a latent state. PPO from the membrane fraction showed no diphenolase activity unless it was activated by trypsin or sodium dodecyl sulfate (SDS). The kinetics of the activation process of latent PPO by trypsin was studied and the specific rate constant of active PPO formation, k 3 , showed a value of 0.03 s −1 . The protease-activated form showed a pH optimum (6.5) and kinetic properties identical to those of the SDS-activated enzyme. Evidence is provided for the existence of a common peptide responsible for the regulation of the activity of the enzyme by both proteolysis and SDS detergent. Formation of the active proteolyzate was followed by spectroscopic measurements, Western blotting and partially denaturing SDS-PAGE.