A Fos-Jun element in the first intron of an α2u-globulin gene

The hepatic expression of the _2u gene family is controlled by a variety of hormones including steroids, growth hormone and insulin. The mechanisms by which these hormones affect -globulin expression are only partially understood. Recently we isolated and characterized clone RAP 01, an _2u-globulin gene expressed in the liver. In preliminary experiments we noted that partial hepatectomy, a procedure which results in a sharp rise in the level of the oncoproteins c-Fos and c-Jun, also causes a transient induction of the messenger RNA corresponding to clone RAP 01. Using the DNAseI footprinting technique we were able to show that this clone contains a TPA (phorbol 12-myristate 13-acetate)-responsive element (TRE) in its first intron. This element (denoted as element X) is identical to the consensus AP-1 binding site (TGACTCAG) and is protected by rat liver nuclear extracts as well as by purified c-Jun. Gel retardation experiments show that an oligonucleotide containing the TRE consensus sequence competes for binding of liver nuclear proteins to element X and that antibodies directed against the M₂ peptide of the mouse Fos protein or the PEP-2 peptide of Jun prevent the formation of specific complexes with the same element. Moreover, element X functions as a TRE in transfected BWTG₃ hepatoma cells treated with TPA. Co-transfection withfos andjun expression vectors mimics the effects of TPA suggesting that AP-1 is in fact the mediator of the observed response. It is concluded that the first intron of RAP 01 contains a functional Fos-Jun element.     

Helix induction potential of N-terminal α-methyl, α-amino acids

Summary A series of longer analogues of the C-peptide of RNAse A has been synthesized with the aim of assessing the helix induction potential in water of α-methyl, α-amino acids at the N-terminus of the chain. The circular dichroism data indicate that one isovaline residue is effective in increasing the helix content of the 13-residue peptide by about 7%.     

Near-critical phenomena in intracellular metabolite pools

The supply and consumption of metabolites in living cells are catalyzed by enzymes. Here we consider two of the simplest schemes where one substrate is eliminated through Michaelis-Menten kinetics, and where two types of substrates are joined together by an enzyme. It is demonstrated how steady-state substrate concentrations can change ultrasensitively in response to changes in their supply rates and how this is coupled to slow relaxation back to steady state after a perturbation. In the one-substrate system, such near-critical behavior occurs when the supply rate approaches the maximal elimination rate, and in the two-substrate system it occurs when the rates of substrate supply are almost balanced. As systems that operate near criticality tend to display large random fluctuations, we also carried out a stochastic analysis using analytical approximations of master equations and compared the results with molecular-level Monte Carlo simulations. It was found that the significance of random fluctuations was directly coupled to the steady-state sensitivity and that the two substrates can fluctuate greatly because they are anticorrelated in such a way that the product formation rate displays only small variation. Basic relations are highlighted and biological implications are discussed.     

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.     

Outer membrane protein A deficient Escherichia coli activates neutrophils to produce superoxide and shows increased susceptibility to antibacterial peptides

The outer membrane protein A (OmpA) of Gram-negative bacteria has been ascribed multiple functions including maintenance of structural membrane integrity and porin activity. OmpA has also been implicated in various host defense processes in that it contributes to bacterial serum resistance and activates certain immune cells. Recently, OmpA was shown to be the molecular target for neutrophil elastase (NE), and Escherichia coli mutants lacking OmpA were resistant to the bactericidal effects of NE. In addition to NE, neutrophils use a variety of other antibacterial effector molecules such as oxygen radicals and bactericidal peptides or proteins. The aim of this study was to investigate the role of E. coli OmpA regarding susceptibility to other neutrophil-derived defense systems. We found that OmpA-deficient (OmpA(-)), but not wild-type isogenic, E. coli activated human neutrophils to produce oxygen radicals intracellularly. This activation was found to require an intact neutrophil cytoskeleton but was independent of bacterial phagocytosis. Furthermore, we found that the OmpA(-) strain was more susceptible to membrane-acting bactericidal peptides than the wild-type strain, although the susceptibility to different oxygen radicals was independent of the presence of OmpA. Taken together, these data suggest an important role for OmpA in the context of bacteria vs. host interactions.     

Characterization of Interactions between the Mammalian Polycomb-Group Proteins Enx1/EZH2 and EED Suggests the Existence of Different Mammalian Polycomb-Group Protein Complexes

In Drosophila melanogaster, the Polycomb-group (PcG) and trithorax-group (trxG) genes have been identified as repressors and activators, respectively, of gene expression. Both groups of genes are required for the stable transmission of gene expression patterns to progeny cells throughout development. Several lines of evidence suggest a functional interaction between the PcG and trxG proteins. For example, genetic evidence indicates that the enhancer of zeste [E(z)] gene can be considered both a PcG and a trxG gene. To better understand the molecular interactions in which the E(z) protein is involved, we performed a two-hybrid screen with Enx1/EZH2, a mammalian homolog of E(z), as the target. We report the identification of the human EED protein, which interacts with Enx1/EZH2. EED is the human homolog of eed, a murine PcG gene which has extensive homology with the Drosophila PcG gene extra sex combs (esc). Enx1/EZH2 and EED coimmunoprecipitate, indicating that they also interact in vivo. However, Enx1/EZH2 and EED do not coimmunoprecipitate with other human PcG proteins, such as HPC2 and BMI1. Furthermore, unlike HPC2 and BMI1, which colocalize in nuclear domains of U-2 OS osteosarcoma cells, Enx1/EZH2 and EED do not colocalize with HPC2 or BMI1. Our findings indicate that Enx1/EZH2 and EED are members of a class of PcG proteins that is distinct from previously described human PcG proteins.In Drosophila melanogaster, the genes of the Polycomb group (PcG) and trithorax group (trxG) are part of a cellular memory system, which is responsible for the stable inheritance of gene activity. The PcG and trxG genes have been identified in Drosophila as repressors (PcG) (18, 22, 27, 28, 38) and activators (trxG) (20, 21), respectively, of homeotic gene activity. PcG and trxG genes were originally found in Drosophila, but mammalian homologs have also been identified and appear to function like their Drosophila homologs (reviewed in reference 37). It has been proposed that PcG proteins repress gene expression through the formation of multimeric protein complexes. We have recently shown that the human PcG proteins HPH1 and HPH2 coimmunoprecipitate, cofractionate, and colocalize in nuclear domains with the human PcG proteins BMI1 (2, 12, 33) and HPC2, a recently identified, novel human Polycomb protein (33, 34). Furthermore, we have found that the human RING1 protein coimmunoprecipitates and colocalizes with HPC2 and other PcG proteins, indicating that RING1 is associated with, or is part of, the mammalian PcG complex (33, 35). These results indicate that mammalian PcG proteins form a multimeric protein complex. This observation is in agreement with observations that different PcG proteins, including Pc, bind in overlapping patterns on polytene chromosomes in Drosophila salivary gland cells (4, 10, 29).Interestingly, also the trithorax gene product trx colocalizes with Drosophila PcG proteins at many sites on polytene chromosomes (6, 24). Even more strikingly, binding of the trx protein has been mapped to small DNA fragments that also contain binding sites for PcG proteins, the Polycomb response elements (5, 6). This finding is further substantiated by the observation that GAGA factor, the gene product of the trxG gene trithorax-like (Trl) (13), colocalizes with Pc protein within the close vicinity of a Polycomb response element (41). Furthermore, the PcG gene Enhancer of zeste [E(z)] contains a domain with sequence homology with the activator protein trx (17). This observation is in agreement with genetic data which indicate that E(z) can be considered both a PcG gene and a trxG gene (26). Double mutations of E(z) and trxG genes result in homeotic phenotypes which are similar to the homeotic phenotypes which are also observed in double mutants of trxG genes (26). Finally, polytene chromosome binding of the trx protein is strongly reduced in homozygous E(z) mutants (4), and vice versa, polytene chromosome binding of the E(z) protein is reduced in trx mutants (24). These data suggest functional interactions between activators (trxG proteins) and repressors (PcG proteins) that are important for their mode of action.To start to investigate these puzzling features of the E(z) gene product, we used the two-hybrid system (8, 9) in order to identify proteins that interact with a mammalian homolog of E(z), the Enx1/EZH2 protein (15, 16). Here, we report the identification of the human EED protein, which interacts with Enx1/EZH2. EED is the human homolog of eed, a murine PcG gene (7, 36) which has extensive homology with the Drosophila PcG gene extra sex combs (esc) (14, 32, 39). Whereas Enx1/EZH2 and EED coimmunoprecipitate, they neither coimmunoprecipitate nor colocalize with other human PcG proteins, such as HPC2 and BMI1. Our findings indicate that both Enx1/EZH2 and EED form a class of mammalian PcG proteins that is distinct from previously described human PcG proteins.     

First partial three-dimensional model of human monoamine oxidase A

A survey of the major known structural aspects of monoamine oxidase (MAO) is given and a first partial model of human MAO A is presented. This 3D model has been established using secondary structure predictions and fold recognition methods. It shows two α/β domains (the FAD-binding N-terminal and central domains) and an α+β domain. The C-terminal region is predicted to be responsible for anchoring the protein into the mitochondrial membrane and was not modeled. The covalent binding of the flavin cofactor to a cysteine residue is well predicted. The model is validated with experimental data from the literature and should be useful in designing new experimental studies (site-directed mutagenesis, chemical modification, specific antibodies). This first step towards the 3D structure of monoamine oxidase should contribute to a better understanding of the mechanisms of action and inhibition of this drug target in the treatment of clinical depression. Proteins 32:97–110, 1998. © 1998 Wiley-Liss, Inc.     

Detection of Pseudomonas aeruginosa in sputum headspace through volatile organic compound analysis

Introduction

Chronic pulmonary infection is the hallmark of Cystic Fibrosis lung disease. Searching for faster and easier screening may lead to faster diagnosis and treatment of Pseudomonas aeruginosa (P. aeruginosa). Our aim was to analyze and build a model to predict the presence of P. aeruginosa in sputa.

Methods

Sputa from 28 bronchiectatic patients were used for bacterial culturing and analysis of volatile compounds by gas chromatography–mass spectrometry. Data analysis and model building were done by Partial Least Squares Regression Discriminant analysis (PLS-DA). Two analysis were performed: one comparing P. aeruginosa positive with negative cultures at study visit (PA model) and one comparing chronic colonization according to the Leeds criteria with P. aeruginosa negative patients (PACC model).

Results

The PA model prediction of P. aeruginosa presence was rather poor, with a high number of false positives and false negatives. On the other hand, the PACC model was stable and explained chronic P. aeruginosa presence for 95% with 4 PLS-DA factors, with a sensitivity of 100%, a positive predictive value of 86% and a negative predictive value of 100%.

Conclusion

Our study shows the potential for building a prediction model for the presence of chronic P. aeruginosa based on volatiles from sputum.     

Unusual lectin-binding properties of a herpes simplex virus type 1-specific glycoprotein

Lysates from herpes simplex virus type 1-infected cells were subjected to affinity chromatography on soybean and Helix pomatia lectins. One of the virus-specified glycoproteins, probably the herpes simplex virus type 1-specific gC glycoprotein, bound to the lectins and was eluted with N-acetylgalactosamine. The affinity chromatography permitted a high degree of purification of the type-specific glycoprotein with respect to both host cell components and other viral glycoproteins. The lectin affinity pattern of this glycoprotein indicates the presence of a terminal alpha-N-acetylgalactosamine in an oligosaccharide, a finding not reported previously for glycoproteins of enveloped viruses.