Christos A. Panagiotidis

Interactions of the antizyme AtoC with regulatory elements of the Escherichia coli atoDAEB operon

AtoC has a dual function as both an antizyme, the posttranslational inhibitor of polyamine biosynthetic enzymes, and the transcriptional regulator of genes involved in short-chain fatty acid catabolism (the atoDAEB operon). We have previously shown that AtoC is the response regulator of the AtoS-AtoC two-component signal transduction system that activates atoDAEB when Escherichia coli is exposed to acetoacetate. Here, we show that the same cis elements control both promoter inducibility and AtoC binding. Chromatin immunoprecipitation experiments confirmed the acetoacetate-inducible binding of AtoC to the predicted DNA region in vivo. DNase I protection footprinting analysis revealed that AtoC binds two 20-bp stretches, constituting an inverted palindrome, that are located at -146 to -107 relative to the transcription initiation site. Analyses of promoter mutants obtained by in vitro chemical mutagenesis of the atoDAEB promoter verified both the importance of AtoC binding for the inducibility of the promoter by acetoacetate and the sigma54 dependence of atoDAEB expression. The integration host factor was also identified as a critical component of the AtoC-mediated induction of atoDAEB.

Regulation of polyamine biosynthesis by antizyme and some recent developments relating the induction of polyamine biosynthesis to cell growth: Review

This review considers the role of antizyme, of amino acids and of protein synthesis in the regulation of polyamine biosynthesis.The ornithine decarboxylase of eukaryotic ceils and ofEscherichia coli coli can be non-competitively inhibited by proteins, termed antizymes, which are induced by di-and poly- amines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. c o l i antizyme and the rat liver antizyme cross react and inhibit each others biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution.Evidence for the physiological role of the antizyme in mammalian cells rests upon its identification in normal uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and antizyme in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 x 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein that specifically binds to the antizyme of an ornithine decarboxylase-antizyme complex and liberates free ornithine decarboxylase from the complex. In B. coli , in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by arginine decarboxylase, three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor lysine decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins S20/L26 and L34, respectively. The relationship of the acidic antizyme to other known B. coli proteins remains to be determined.

Superinduction of cytosolic and chromatin-bound ornithine decarboxylase activities of germinating barley seeds by actinomycin D

Putrescine in plant cells is formed either from Larginine by L-arginine decarboxylase (EC 4.11.19, ADC) or directly from L-ornithine by L-ornithine decarboxylase (EC 4.11.17, ODC) [1-5]. The contribution of ODC to the formation of polyamines in plants was claimed to be insignificant, since ODC activity in most plant tissues was found to be much lower than that of ADC [6]. The only welldocumented work on ODC activity in plant cells is that in [4] on rapidly proliferating plant cells. Investigations to now on plant ODC were performed in the 10000 × g supernatant of plant tissue homogenates based on the assumption that ODC is a cytosolic enzyme. Here, evidence is presented that in barley seeds germinated for > 90 h, ODC activity is located mainly in the nucleus, tightly bound to chromatin, although the cytosol also possesses considerable activity. Both activities are superinduced when seed germination takes place in the presence of gibberellic acid and actinomycin D.

Bluetongue virus diagnosis of clinical cases by a duplex reverse transcription-PCR: a comparison with conventional methods

A duplex reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of bluetongue virus (BTV) in clinical samples was developed. This assay, which detects the highly conserved S10 region of BTV, was assessed for sensitivity and application as a rapid and dependable diagnostic tool by comparison with standard assays of virus detection, such as virus isolation in embryonated chicken eggs and cell culture. Simultaneous detection of BTV and host beta-actin RNAs minimizes the possibility of false negative results. The sensitivity of the assay was found to be equal to five cell culture infectious dose (CCID(50)) units and its specificity was confirmed as no RT-PCR product was detected with RNAs from two closely related orbiviruses, i.e. epizootic haemorrhagic disease virus (serotypes 1, 2 and 318) and African horse sickness virus, serotype 9, or RNAs from uninfected BHK-21 cells and blood samples from uninfected sheep or goats. In this study, 36 blood samples from naturally infected mixed flocks of sheep and goats were examined. Seventeen animals were identified as BTV-positive by RT-PCR, whereas only 13 were found positive by virus isolation in embryonated chicken eggs and nine by cell culture assays. These results indicate that the duplex RT-PCR could be a useful technique for monitoring BTV infection in the field.

Chaperone-fusion expression plasmid vectors for improved solubility of recombinant proteins in Escherichia coli

The enteric bacterium Escherichia coli is the most extensively used prokaryotic organism for production of proteins of therapeutic or commercial interest. However, it is common that heterologous over-expressed recombinant proteins fail to properly fold resulting in formation of insoluble aggregates known as inclusion bodies. Complex systems have been developed that employ simultaneous over-expression of chaperone proteins to aid proper folding and solubility during bacterial expression. Here we describe a simple method whereby a protein of interest, when fused in frame to the E. coli chaperones DnaK or GroEL, is readily expressed in large amounts in a soluble form. This system was tested using expression of the mouse prion protein PrP, which is normally insoluble in bacteria. We show that while in trans over-expression of the chaperone DnaK failed to alter partitioning of PrP from the insoluble inclusion body fraction to the soluble cytosol, expression of a DnaK-PrP fusion protein yielded large amounts of soluble protein. Similar results were achieved with a fragment of insoluble Varicella Zoster virus protein ORF21p. In theory this approach could be applied to any protein that partitions with inclusion bodies to render it soluble for production in E. coli.

Intracellular Transport of Varicella-Zoster Glycoproteins

Previous observations have established that varicella-zoster virus (VZV) is enveloped in the trans-Golgi network (TGN) in cultures infected with VZV and that the glycoprotein gE is targeted to the TGN by a signal sequence (AYRV) and an acidic TGN signal patch in its cytosolic domain. Neither sequence is present in other VZV glycoproteins. Like gE, gI was targeted to the TGN when it was expressed in transfected cells, suggesting that gI also contains TGN targeting information (colocalized with gE and the AP-1 adaptin complex). In contrast, gB, gC, gH, and gL immunoreactivities were not detected in the TGN when they were expressed individually in transfected cells. In VZV-infected cells, gE, gI, gH, and gL were all concentrated in the TGN. Since VZV glycoproteins that lack targeting sequences (gB, gC, gH, and gL) concentrated in the TGN of infected cells, it is proposed that gE and gI, which have such sequences, serve as navigator glycoproteins, forming complexes that direct the signal-deficient glycoproteins to the TGN.

Effect of polyamines and synthetic polyamine-analogues on the expression of antizyme (AtoC) and its regulatory genes.

BackgroundIn bacteria, the biosynthesis of polyamines is modulated at the level of transcription as well as post-translationally. Antizyme (Az) has long been identified as a non-competitive protein inhibitor of polyamine biosynthesis in E. coli. Az was also revealed to be the product of the atoC gene. AtoC is the response regulator of the AtoS-AtoC two-component system and it functions as the positive transcriptional regulator of the atoDAEB operon genes, encoding enzymes involved in short chain fatty acid metabolism. The antizyme is referred to as AtoC/Az, to indicate its dual function as both a transcriptional and post-translational regulator.ResultsThe roles of polyamines on the transcription of atoS and atoC genes as well as that of atoDAEB(ato) operon were studied. Polyamine-mediated induction was tested both in atoSC positive and negative E. coli backgrounds by using β-galactosidase reporter constructs carrying the appropriate promoters patoDAEB, patoS, patoC. In addition, a selection of synthetic polyamine analogues have been synthesized and tested for their effectiveness in inducing the expression of atoC/Az, the product of which plays a pivotal role in the feedback inhibition of putrescine biosynthesis and the transcriptional regulation of the ato operon. The effects of these compounds were also determined on the ato operon expression. The polyamine analogues were also tested for their effect on the activity of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis and on the growth of polyamine-deficient E. coli.ConclusionPolyamines, which have been reported to induce the protein levels of AtoC/Az in E. coli, act at the transcriptional level, since they cause activation of the atoC transcription. In addition, a series of polyamine analogues were studied on the transcription of atoC gene and ODC activity.

Relationship of the expression of the S20 and L34 ribosomal proteins to polyamine biosynthesis in Escherichia coli

Polyamine biosynthesis in Escherichia coli is regulated transcriptionally and post-translationally. Antizyme and ribosomal proteins S20 and L34 participate in post-translational inhibition of the polyamine biosynthetic enzymes ornithine and arginine decarboxylase. The aim of the present study was to investigate the significance of S20 and L34 in polyamine regulation in vivo. In vivo overexpression of S20 and L34 lowered the activities of ornithine and arginine decarboxylases and decreased total polyamine production. The levels of cadaverine, a related diamine whose synthesis is not regulated by S20 and L34, did not decrease but increased. The diminished ornithine and arginine decarboxylase activities are shown to result from reversible post-translational inhibition since the enzymes could be reactivated to normal levels upon titration of the inhibitors. The effects were specific as overexpression of eight other ribosomal proteins had no influence. Overexpression of ornithine decarboxylase results in elevated polyamine production and it increases S20 and L34 levels but not those of other ribosomal proteins. Ornithine depletion decreases S20 and L34 to normal levels in the ornithine decarboxylase overproducing cells. Immunoprecipitation experiments coupled with immunoblots indicated that ornithine and arginine decarboxylases physically interact with S20 and L34. This study shows that ribosomal proteins S20 and L34 can inhibit ornithine and arginine decarboxylases and polyamine biosynthesis in vivo. It is concluded that, unlike other basic ribosomal proteins and polycationic compounds which inhibit the activities of these enzymes only in vitro, S20 and L34 are biologically relevant in the regulation of the polyamine biosynthetic pathway.

Barley β-glucosidase: Expression during seed germination and maturation and partial amino acids sequences

Abstract Unlike most of the hydrolytic enzymes that participate in endosperm mobilization, β-glucosidase of barley ( Hordeum vulgare ) seeds does not increase during germination, even in the presence of exogenously added gibberellic acid. However, the germination process affects the physical properties of β-glucosidase in terms of charge and apparent molecular weight. Analysis of developing barley grains shows that the enzyme is synthesized two weeks before maturation and is stored in the endosperm of the dry dormat seed. Partial amino acid sequencing of the purified β-glucosidase demonstrates significant similarity between the barley enzyme and β-glycosidases that belong to family 1 of glycosyl hydrolases.

Varicella-zoster virus proteins in skin lesions: implications for a novel role of ORF29p in chickenpox.

ABSTRACT Skin biopsy samples from varicella-zoster virus (VZV)-infected patients examined by immunohistochemistry demonstrated VZV replication in nonepithelial cell types. ORF29p, a nonstructural nuclear protein, was found in nerves of two of six patients with chickenpox. In tissue culture, ORF29p was secreted by VZV-infected fibroblasts. Extracellular ORF29p can be taken up through endocytosis by human neurons, implying a novel role for this protein in pathogenesis.