bluuebunny/crossref_metadata_2025 huggingface dataset:
The Abitinian Martyrs and the Outbreak of the Donatist Schism
Authors: DEARN ALAN
Published: May 2004

It is often claimed that the story of the Abitinian martyrs provides evidence relevant to the outbreak of the Donatist schism. However, this interpretation of the text rests upon a number of false assumptions regarding its manuscript tradition and the celebrity of the martyrs it commemorates. Rather than being written in the early fourth century, the text in its extant form was probably written after the Council of Carthage in 411, and perhaps in response to it. It thus does not cast light upon the outbreak of the schism, but rather on the way in which the events of the early fourth century were polemically reinterpreted at a much later date.
Bacteriophage capsids: Tough nanoshells with complex elastic properties
Authors: Ivanovska I. L. , de Pablo P. J. , Ibarra B. , Sgalari G. , MacKintosh F. C. , Carrascosa J. L. , Schmidt C. F. , Wuite G. J. L.
Published: May 2004

The shell of bacteriophages protects the viral DNA during host-to-host transfer and serves as a high-pressure container storing energy for DNA injection into a host bacterium. Here, we probe the mechanical properties of nanometer-sized bacteriophage φ29 shells by applying point forces. We show that empty shells withstand nanonewton forces while being indented up to 30% of their height. The elastic response varies across the surface, reflecting the arrangement of shell proteins. The measured Young's modulus (≈1.8 GPa) is comparable with that of hard plastic. We also observe fatigue and breakage of capsids after probing them repetitively. These results illustrate the mechanoprotection that viral shells provide and also suggest design principles for nanotechnology.
The Candidate Neuroprotective Agent Artemin Induces Autonomic Neural Dysplasia without Preventing Peripheral Nerve Dysfunction
Authors: Bolon Brad , Jing Shuqian , Asuncion Frank , Scully Sheila , Pisegna Marlese , Van Gwyneth Y. , Zheng Hu  , Yan Bin Yu  , Min Hosung , Wild Ken , Rosenfeld Robert D. , Tarpley John , Carnahan Josette , Duryea Diane , Hill Dave , Kaufman Steve , Yan Xiao-Qiang , Juan Todd , Christensen Kathy , Mccabe James , Simonet W. Scott
Published: May 2004

Artemin (ART) signals through the GFR α—3/RET receptor complex to support sympathetic neuron development. Here we show that ART also influences autonomic elements in adrenal medulla and enteric and pelvic ganglia. Transgenic mice over-expressing Art throughout development exhibited systemic autonomic neural lesions including fusion of adrenal medullae with adjacent paraganglia, adrenal medullary dysplasia, and marked enlargement of sympathetic (superior cervical and sympathetic chain ganglia) and parasympathetic (enteric, pelvic) ganglia. Changes began by gestational day 12.5 and formed progressively larger masses during adulthood. Art supplementation in wild type adult mice by administering recombinant protein or an Art-bearing retroviral vector resulted in hyperplasia or neuronal metaplasia at the adrenal corticomedullary junction. Expression data revealed that Gfr α—3 is expressed during development in the adrenal medulla, sensory and autonomic ganglia and their projections, while Art is found in contiguous mesenchymal domains (especially skeleton) and in certain nerves. Intrathecal Art therapy did not reduce hypalgesia in rats following nerve ligation. These data (1) confirm that ART acts as a differentiation factor for autonomic (chiefly sympathoadrenal but also parasympathetic) neurons, (2) suggest a role for ART overexpression in the genesis of pheochromocytomas and paragangliomas, and (3) indicate that ART is not a suitable therapy for peripheral neuropathy.
Myopathy Related to Administration of a Cationic Amphiphilic Drug and the Use of Multidose Drug Distribution Analysis to Predict its Occurrence
Authors: Vonderfecht Steven L. , Stone Marjorie L. , Eversole Robert R. , Yancey Mark F. , Schuette Margaret R. , Duncan Barbara A. , Ware Joseph A.
Published: May 2004

Many cationic amphiphilic (phospholipidosis-inducing) drugs (CADs) accumulate in tissues following repeated dosing in preclinical models, and this is sometimes associated with dose-limiting toxicities. Plasma drug levels cannot be used to estimate tissue accumulation of CADs since it occurs in tissues despite stabilization of plasma levels. Severe myopathy was found in skeletal muscles of rats during the initial safety evaluation of a dopamine D3 receptor antagonist, PNU-177864, and was associated with phospholipidosis in numerous tissues. The myopathy was observed only when plasma levels of PNU-177864 remained essentially constant throughout the 24-hour dosing period. A repeat dose drug distribution study using whole body autoradiography demonstrated that drug-related material did not accumulate in skeletal muscle or other tissues following repeated doses at levels considered within the therapeutic range and showing toxicokinetic profiles acceptable for further development. These observations provided support for the continued development of and longer-term toxicity studies with this candidate compound.
Electron Microscopy of Wet Tissues: A Case Study in Renal Pathology
Authors: Nyska Abraham , Cummings Connie A. , Vainshtein Anya , Nadler Jonathan , Ezov Nathan , Grunfeld Yona , Gileadi Opher , Behar Vered
Published: May 2004

In this report we introduce wet-tissue scanning electron microscopy, a novel technique for direct imaging of wet tissue samples using backscattered electrons. Samples placed in sealed capsules are imaged through a resilient, electron-transparent membrane. The contrast of the imaged samples may be enhanced by chemical staining. The samples several millimeters thick and imaged without sectioning, makes this technique suitable for rapid analysis of tissue specimens. We applied this technique to D-limonene-induced nephropathy where accumulation of hyaline protein droplets is induced in proximal and distal convoluted tubules of the kidney. Images obtained by scanning electron microscopy of hydrated kidney specimens exhibited superior resolution, contrast, and magnification compared with those obtained by conventional light microscopy of paraffin sections. The electron micrographs can be obtained within an hour of tissue removal, whereas preparation for light microscopy requires at least 1 day. These advantages of the wet scanning electron microscopy technique indicate its potential utility in a wide range of applications in histopathology and toxicology.
Diversity and Evolution of Mitochondrial RNA Editing Systems
Authors: Gray Michael
Published: May 2004

'RNA editing' describes the programmed alteration of the nucleotide sequence of an RNA species, relative to the sequence of the encoding DNA. The phenomenon encompasses two generic patterns of nucleotide change, 'insertion/deletion' and 'substitution', defined on the basis of whether the sequence of the edited RNA is colinear with the DNA sequence that encodes it. RNA editing is mediated by a variety of pathways that are mechanistically and evolutionarily unrelated. Messenger, ribosomal, transfer and viral RNAs all undergo editing in different systems, but well‐documented cases of this phenomenon have so far been described only in eukaryotes, and most often in mitochondria. Editing of mRNA changes the identity of encoded amino acids and may create translation initiation and termination codons. The existence of RNA editing violates one of the long‐accepted tenets of genetic information flow, namely, that the amino acid sequence of a protein can be directly predicted from the corresponding gene sequence. Particular RNA editing systems display a narrow phylogenetic distribution, which argues that such systems are derived within specific eukaryotic lineages, rather than representing traits that ultimately trace to a common ancestor of eukaryotes, or even further back in evolution. The derived nature of RNA editing raises intriguing questions about how and why RNA editing systems arise, and how they become fixed as additional, essential steps in genetic information transfer. IUBMB Life, 55: 227‐233, 2003
DNA Damage Regulation of the RNA Components of the Translational Apparatus: New Biology and Mechanisms
Authors: Schultz Michael
Published: May 2004

It was shown more than 30 years ago that expression of ribosomal (r) RNAs processed from the large precursor rRNA is repressed when eukaryotic cells are exposed to genotoxic stress. More recently it has been found that other RNA components of the translational machinery, the tRNAs and 5S rRNA transcribed by RNA polymerase (pol) III, are also downregulated in cells that have experienced DNA damage. In other words, the DNA damage response involves coordinate repression of genes whose products comprise the heart of the translational machinery. This repression could be due to blockage of polymerase elongation, and indeed this mechanism was originally invoked to explain repression of pol I‐transcribed rRNAs under conditions of genotoxic stress. Recent work however reveals the existence of a DNA damage signaling pathway that directly contributes to downregulation of the pol III and probably the pol I transcription initiation machinery. This pathway involves a highly conserved protein kinase, CK2. Its likely target is the TATA Binding Protein, which in most eukaryotes is required for transcription by both pol I and pol III. Here I consider the implications of these findings for our understanding of the physiology of the DNA damage response, and for the prospect of developing a comprehensive molecular model of how cells cope with genotoxic stress. IUBMB Life, 55: 243‐247, 2003
Protein Insertion into the Inner Membrane of Mitochondria
Authors: Herrmann Johannes , Neupert Walter
Published: May 2004

The inner membrane of mitochondria harbours a large number of polypeptides, many of which have evolved from proteins of the prokaryotic progenitors of mitochondria. The sorting routes on which these proteins are integrated into the mitochondrial inner membrane reflect their phylogenetic origin: Proteins of eukaryotic descent typically reach their destination following arrest of import at the level of the inner membrane. In contrast, many proteins inherited from the prokaryotic progenitor cell are inserted into the inner membrane in an export step following translocation into the matrix. Recently, three different insertion pathways from the matrix into the inner membrane were identified which show considerable parallels to the protein insertion processes in bacteria and chloroplasts. Two of these pathways depend on the related inner membrane proteins Oxa1 and Cox18. A third route is less well defined and depends on the membrane‐associated matrix protein Mba1. IUBMB Life, 55: 219‐225, 2003
Target Selection and Determination of Function in Structural Genomics
Authors: Watson James , Todd Annabel , Bray James , Laskowski Roman , Edwards Aled , Joachimiak Andrzej , Orengo Christine , Thornton Janet
Published: May 2004

The first crucial step in any structural genomics project is the selection and prioritization of target proteins for structure determination. There may be a number of selection criteria to be satisfied, including that the proteins have novel folds, that they be representatives of large families for which no structure is known, and so on. The better the selection at this stage, the greater is the value of the structures obtained at the end of the experimental process. This value can be further enhanced once the protein structures have been solved if the functions of the given proteins can also be determined. Here we describe the methods used at either end of the experimental process: firstly, sensitive sequence comparison techniques for selecting a high‐quality list of target proteins, and secondly the various computational methods that can be applied to the eventual 3D structures to determine the most likely biochemical function of the proteins in question. IUBMB Life, 55: 249‐255, 2003
Mitochondrial DNA Replication: What We Know
Authors: Clayton David
Published: May 2004

Since the isolation and physical characterization of mammalian mitochondrial DNA (mtDNA) over 35 years ago, numerous works have been published that have examined its physical structure and properties, including its mode of replication and transcription. The established replication model posits that leading‐strand replication of mammalian mtDNA begins at closely spaced, defined sites located downstream from a major transcription promoter and proceeds unidirectionally with displacement of the parental leading strand until approximately two‐thirds of the closed circular mtDNA has been copied. As a consequence, the replication fork passes a major origin for lagging‐strand synthesis, exposing it in single‐stranded form. Displacement as a single‐strand is thought to allow the characteristic secondary structure of this origin to occur, thereby permitting initiation of lagging‐strand synthesis. A natural consequence of the separate and distinct locations of the two origins is that the two segregated progeny mtDNA circles are of two types: one a duplex circle with a newly synthesized leading strand and the other a gapped circle with a partial newly synthesized lagging strand. In each case, the final steps of synthesis and ligation result in closed circular mtDNA products. Recently, mammalian mtDNA isolates have been subjected to 2D‐gel electrophoretic analysis in attempts to assign features to mtDNA molecules that, by virtue of their anomalous migration behavior, could infer them to be candidates for replication intermediates. This review will describe the essential features of the historical findings of mammalian mtDNA replication studies and integrate the more recent observations in developing a current model for this process. IUBMB Life, 55: 213‐217, 2003
Parallels in Genome Evolution in Mitochondria and Bacterial Symbionts
Authors: Burger Gertraud , Lang B Franz
Published: May 2004

Mitochondria, the energy‐producing organelles of the eukaryotic cell, originate from an endosymbiotic α‐proteobacterium. These organelles are believed to have arisen only once in evolutionary history, but despite their common ancestry, mitochondrial DNAs vary extensively throughout eukaryotes in genome architecture and gene content. New insights into early mitochondrial genome evolution come from the investigation of primitive mitochondriate eukaryotes, as well as the comparison between mitochondria and intracellular bacterial symbionts. IUBMB Life, 55: 205‐212, 2003
Early Cell Evolution, Eukaryotes, Anoxia, Sulfide, Oxygen, Fungi First (?), and a Tree of Genomes Revisited
Authors: Martin William , Rotte Carmen , Hoffmeister Meike , Theissen Ursula , Gelius‐Dietrich Gabriel , Ahr Simone , Henze Katrin
Published: May 2004

Genomes contain evidence for the history of life and furthermore contain evidence for lateral gene transfer, which was an important part of that history. The geological record also contains evidence for the history of life, and newer findings indicates that the Earth's oceans were largely anoxic and highly sulfidic up until about 0.6 billion years ago. Eukaryotes, which fossil data indicate to have been in existence for at least 1.5 billion years, must have therefore spent much of their evolutionary history in oxygen‐poor and sulfide‐rich environments. Many eukaryotes still inhabit such environments today. Among eukaryotes, organelles also contain evidence for the history of life and have preserved abundant traces of their anaerobic past in the form of energy metabolic pathways. New views of eukaryote phylogeny suggest that fungi may be among the earliest‐branching eukaryotes. From the standpoint of the fungal feeding habit (osmotrophy rather than phagotrophy) and from the standpoint of the diversity in their ATP‐producing pathways, a eukaryotic tree with fungi first would make sense. Because of lateral gene transfer and endosymbiosis, branches in the tree of genomes intermingle and occasionally fuse, but the overall contours of cell history nonetheless seem sketchable and roughly correlateable with geological time. IUBMB Life, 55: 193‐204, 2003
Influences of temperature, salinity and starvation on the motility and chemotactic response of Vibrio anguillarum
Authors: Larsen Marianne H. , Blackburn Nicholas , Larsen Jens L. , Olsen John E.
Published: May 2004

The role of growth factors for the motility and chemotaxis of the fish pathogenVibrio anguillarumwas determined. Cells ofV. anguillarumwere chemotactic to serine in the temperature range 5–25 °C and in 0·8–2·7 % NaCl. The chemotactic response was significantly higher at 25 °C than at 5 or 15 °C. Growth in medium with 1·5 % NaCl gave a higher response than growth with 3 % NaCl; when the salinity of the chemotaxis buffer was raised, the chemotactic response was reduced. The role of starvation was also studied;V. anguillarumshowed a high chemotactic response after starvation for 2 and 8 days. Motility and chemotaxis are important virulence factors for this bacterium. Not only was the ability to perform chemotactic motility maintained after starvation, but also it was shown that starvation does not interfere with the ability of the organism to cause infection in rainbow trout after a bath challenge. The swimming speed was reduced at lower temperatures. Within the range of salinity and starvation studied, the motile cells swam with the same velocity, indicating thatV. anguillarumunder all the examined conditions has a functional flagellum and rotates it with constant speed. Phenamil, a specific inhibitor of Na+-driven flagella, reduced the motility of both starved and non-starved cells ofV. anguillarumindicating that, in both cases, a Na+motive force drives the flagellum.
Ammonium and hydroxylamine uptake and accumulation in Nitrosomonas
Authors: Schmidt Ingo , Look Christiane , Bock Eberhard , Jetten Mike S. M.
Published: May 2004

Starved cells ofNitrosomonas europaeaand further ammonia oxidizers were able to rapidly accumulate ammonium and hydroxylamine to an internal concentration of about 1 and 0·8 M, respectively. In kinetic studies, the uptake/accumulation rates for ammonium [3·1 mmol (g protein)−1min−1] and hydroxylamine [4·39 mmol (g protein)−1min−1] were determined. The uptake and accumulation process of ammonium and hydroxylamine was not coupled to ammonia or hydroxylamine oxidation and nitrite was not produced. In the presence of uncouplers the ammonium accumulation was completely inhibited, indicating an active, membrane-potential-driven transport mechanism. When the external ammonium or hydroxylamine pool was depleted, the internal ammonium and hydroxylamine was consumed within 12 h or 20 min, respectively. The binding of ammonium/ammonia was correlated with an energized membrane system, and hydroxylamine may bind to the hydroxylamine oxidoredutase.
Emergence of phenotypic variants upon mismatch repair disruption in Pseudomonas aeruginosa
Authors: Smania Andrea M. , Segura Ignacio , Pezza Roberto J. , Becerra Cecilia , Albesa Inés , Argaraña Carlos E.
Published: May 2004

MutS is part of the bacterial mismatch repair system that corrects point mutations and small insertions/deletions that fail to be proof-read by DNA polymerase activity. In this work it is shown that the disruption of theP. aeruginosa mutSgene generates the emergence of diverse colony morphologies in contrast with its parental wild-type strain that displayed monomorphic colonies. Interestingly, two of themutSmorphotypes emerged at a high frequency and in a reproducible way and were selected for subsequent characterization. One of them displayed a nearly wild-type morphology while the other notably showed, compared with the wild-type strain, increased production of pyocyanin and pyoverdin, lower excretion of LasB protease and novel motility characteristics, mainly related to swarming. Furthermore, it was reproducibly observed that, after prolonged incubation in liquid culture, the pigmented variant consistently emerged from themutSwild-type-like variant displaying a reproducible event. It is also shown that theseP. aeruginosa mutSmorphotypes not only displayed an increase in the frequency of antibiotic-resistant mutants, as described for clinicalP. aeruginosamutator isolates, but also generated mutants whose antibiotic-resistant levels were higher than those measured from spontaneous resistant mutants derived from wild-type cells. It was also found that both morphotypes showed a decreased cytotoxic capacity compared to the wild-type strain, leading to the emergence of invasive variants. By using mutated versions of a tetracycline resistance gene, themutSmutant showed a 70-fold increase in the reversion frequency of a +1 frameshift mutation with respect to its parental wild-type strain, allowing the suggestion that the phenotypical diversity generated in themutSpopulation could be produced in part by frameshift mutations. Finally, since morphotypical diversification has also been described in clinical isolates, the possibility that thismutSdiversification was related to the high frequency hypermutability observed inP. aeruginosaCF isolates is discussed.
Strains of Bacillus cereus vary in the phenotypic adaptation of their membrane lipid composition in response to low water activity, reduced temperature and growth in rice starch
Authors: Haque Md Anwarul , Russell Nicholas J.
Published: May 2004

The phenotypic adaptation of membrane lipids in seven strains of the food-poisoning bacterium Bacillus cereus, isolated from Bangladeshi rice, is reported in relation to their ability to grow under conditions of low water activity (a
w), reduced temperature and the presence of soluble rice starch. The strains have different membrane phospholipid head-group and fatty acyl compositions, and they display individual differences in their responses to both low a
w and reduced temperature. The extent of the increase in anionic membrane lipids in response to low a
w varies from strain to strain, is solute specific and in one strain does not occur. Growth is stimulated by the presence of soluble rice starch and results in a large rise in the proportion of diphosphatidylglycerol (DPG) at the expense of phosphatidylglycerol (PG), without any change in the proportion of total anionic phospholipids. Growth at 15 °C compared with 37 °C increases the proportions of DPG and phosphatidylethanolamine at the expense of PG. At the lower temperature there are changes in phospholipid fatty acyl composition characteristic of those expected to maintain membrane fluidity, including increases in the amount of total branched fatty acids and the anteiso-/iso-branched ratio, and a decrease in the equivalent chain-length, but there are strain differences in how those changes were achieved. In contrast to some other bacilli, there are persistent large increases in the proportions of unsaturated fatty acyl chains in phospholipids during growth at 15 °C.
Evolution of multi-resistance plasmids in Australian clinical isolates of Escherichia coli
Authors: Sherley Miranda , Gordon David M. , Collignon Peter J.
Published: May 2004

Plasmids allow the movement of genetic material, including antimicrobial resistance genes, between bacterial species and genera. They frequently mediate resistance to multiple antimicrobials and can result in the acquisition by a pathogen of resistance to all or most clinically relevant antimicrobials. Unfortunately, there are still large gaps in our understanding of how new multi-resistance plasmids evolve. Five Australian clinical institutions collaborated in this study of multi-resistance plasmids in clinical isolates ofEscherichia coli. We characterized 72 resistance plasmids in terms of the antimicrobial resistance profile they conferred, their size and their incompatibility group. Restriction fragment length polymorphisms were used to determine the genetic relationships between the plasmids. Relationships between the host cells were determined using multi-locus enzyme electrophoresis. A lack of correlation between the evolutionary history of the host cells and their plasmids suggests that the horizontal transfer of resistance plasmids between strains ofE. coliis common. The resistance plasmids were very diverse, with a wide range of resistance profiles and a lack of discrete evolutionary lineages. Multi-resistance plasmids did not evolve via the co-integrative capture of smaller resistance plasmids; rather, the roles of recombination and the horizontal movement of mobile genetic elements appeared to be most important.
The signal peptide sequence of a lytic transglycosylase of Neisseria meningitidis is involved in regulation of gene expression
Authors: Serruto Davide , Galeotti Cesira L.
Published: May 2004

The 60 nucleotides encoding the signal peptide of theNeisseria meningitidismembrane-bound lytic transglycosylase (MltA) homologue GNA33 were found to exert a negative regulatory effect on expression ofGNA33from either a T7- or a Plac-driven system inEscherichia coli. Down-regulation was observed to occur at the transcriptional/post-transcriptional level and could possibly be ascribed to the formation of a stem–loop secondary structure within the signal peptide sequence. Slowing down the transcription rate through inhibition/titration of the RNA polymerase resulted in a considerable increase in mRNA accumulation, suggesting that a better coupling of translation to transcription would impede the formation of the putative secondary structure. Screening of synonymous mutations in the signal peptide sequence that showed high-level expression of an in-frame fusion to a reporter resulted in the isolation of several deletion mutants lacking most of the sequence participating in the putative secondary structure. Interestingly, the increase in the steady-state mRNA level observed in deletion mutants was higher, reaching a 300-fold increment, than that found in substitution mutants. Our results support the hypothesis that the rate of transcription controls the formation of a secondary structure in the region of theGNA33transcript corresponding to the signal peptide sequence and this, when formed, negatively regulates expression.
The plasma membrane of microaerophilic protists: oxidative and nitrosative stress
Authors: Lloyd D. , Harris J. C. , Biagini G. A. , Hughes M. R. , Maroulis S. , Bernard C. , Wadley R. B. , Edwards M. R.
Published: May 2004

The trans-plasma-membrane electrochemical potential of microaerophilic protists was monitored by the use of voltage-sensitive charged lipophilic fluorophores; of the many available probes, the anionic oxonol dye bis(1,3-dibarbituric acid)-trimethine oxonol [DiBAC4(3)] is an example of one which has been successfully employed using fluorescence microscopy, confocal laser-scanning microscopy and flow cytometry. Several microaerophilic protists have been investigated with this dye; these were Giardia intestinalis, Trichomonas vaginalis, Tritrichomonas foetus, Hexamita inflata and Mastigamoeba punctachora. Under conditions where they exhibit normal vitality, these organisms exclude DiBAC4(3) by virtue of their maintenance of a plasma-membrane potential (negative inside). Uptake of the fluorophore is indicative of disturbance to this membrane (i.e. by inhibition of pump/leak balance, blockage of channels or generation of ionic leaks), and is indicative of metabolic perturbation or environmental stress. Here, it is shown that oxidative or nitrosative stress depolarizes the plasma membranes of the aforementioned O2-sensitive organisms and allows DiBAC4(3) influx. Oxonol uptake thereby provides a sensitive and early indication of plasma-membrane perturbation by agents that may lead to cytotoxicity and eventually to cell death by necrotic or apoptotic pathways.
Role of homoserine and threonine pathway intermediates as precursors for the biosynthesis of aminoethoxyvinylglycine in Streptomyces sp. NRRL 5331
Authors: Fernández Mónica , Cuadrado Yolanda , Aparicio Jesús F. , Martín Juan F.
Published: May 2004

The geneshom,thrBandthrC, encoding homoserine dehydrogenase, homoserine kinase (HK) and threonine synthase, respectively, involved in the last steps of threonine biosynthesis, have been studied inStreptomycessp. NRRL 5331, the producer of the ethylene synthetase inhibitor aminoethoxyvinylglycine (AVG), in order to determine their role in the biosynthesis of AVG. Different null mutants were obtained by plasmid-mediated disruption of each of the three genes.thrCgene disruption had no effect on AVG production, while the disruption ofthrBblocked HK activity and substantially reduced the yield of this metabolite, probably due to the accumulation of homoserine and/or methionine which have a negative effect on AVG biosynthesis. Disruption ofhom(thrA) completely blocked AVG biosynthesis, indicating that homoserine lies at the branching point of the aspartic-acid-derived biosynthetic route that leads to AVG. The four carbon atoms of the vinylglycine moiety of AVG derive, therefore, from homoserine.
Proteome analysis of Streptococcus mutans metabolic phenotype during acid tolerance
Authors: Len Alice C. L. , Harty Derek W. S. , Jacques Nicholas A.
Published: May 2004

Two-dimensional gel electrophoretic analysis of the proteome ofStreptococcus mutansgrown at a steady state in a glucose-limited anaerobic continuous culture revealed a number of proteins that were differentially expressed when the growth pH was lowered from pH 7·0 to pH 5·0. Changes in the expression of metabolic proteins were generally limited to three biochemical pathways: glycolysis, alternative acid production and branched-chain amino acid biosynthesis. The relative level of expression of protein spots representing all of the enzymes associated with the Embden–Meyerhof–Parnas pathway, and all but one of the enzymes involved in the major alternative acid fermentation pathways ofS. mutans, was identified and measured. Proteome data, in conjunction with end-product and cell-yield analyses, were consistent with a phenotypic change that allowedS. mutansto proliferate at low pH by expending energy to extrude excess H+from the cell, while minimizing the detrimental effects that result from the uncoupling of carbon flux from catabolism and the consequent imbalance in NADH and pyruvate production. The changes in enzyme levels were consistent with a reduction in the formation of the strongest acid, formic acid, which was a consequence of the diversion of pyruvate to both lactate and branched-chain amino acid production whenS. mutanswas cultivated in an acidic environment.
Polyamine metabolism in a member of the phylum Microspora (Encephalitozoon cuniculi): effects of polyamine analogues
Authors: Bacchi Cyrus J. , Rattendi Donna , Faciane Evangeline , Yarlett Nigel , Weiss Louis M. , Frydman Benjamin , Woster Patrick , Wei Benjamin , Marton Laurence J. , Wittner Murray
Published: May 2004

The uptake, biosynthesis and catabolism of polyamines in the microsporidian parasiteEncephalitozoon cuniculiare detailed with reference to the effects of oligoamine and arylamine analogues of polyamines.Enc. cuniculi, an intracellular parasite of mammalian cells, has both biosynthetic and catabolic enzymes of polyamine metabolism, as demonstrated in cell-free extracts of mature spores. The uptake of polyamines was measured in immature, pre-emergent spores isolated from host cells by Percoll gradient. Spermine was rapidly taken up and metabolized to spermidine and an unknown, possibly acetamidopropanal, by spermidine/spermineN1-acetyltransferase (SSAT) and polyamine oxidase (PAO). Most of the spermidine and the unknown product were found in the cell incubation medium, indicating they were released from the cell. bis(Ethyl) oligoamine analogues of polyamines, such as SL-11144 and SL-11158, as well as arylamine analogues [BW-1, a bis(phenylbenzyl) 3-7-3 analogue] blocked uptake and interconversion of spermine at micromolar levels and, in the case of BW-1, acted as substrate for PAO. TheEnc. cuniculiPAO activity differed from that found in mammalian cells with respect to pH optimum, substrate specificity and sensitivity to known PAO inhibitors. SL-11158 inhibited SSAT activity with a mixed type of inhibition in which the analogue had a 70-fold higher affinity for the enzyme than the natural substrate, spermine. The interest inEnc. cuniculipolyamine metabolism and the biochemical effects of these polyamine analogues is warranted since they cure model infections ofEnc. cuniculiin mice and are potential candidates for human clinical trials.
Molecular typing of Pneumocystis jirovecii found in formalin-fixed paraffin-embedded lung tissue sections from sudden infant death victims
Authors: Chabé M. , Vargas S. L. , Eyzaguirre I. , Aliouat E. M. , Follet-Dumoulin A. , Creusy C. , Fleurisse L. , Recourt C. , Camus D. , Dei-Cas E. , Durand-Joly I.
Published: May 2004

Previous studies have provided histological evidence of an association between primaryPneumocystisinfection and sudden infant death syndrome (SIDS). The aim of this work was to determine the species of clusteredPneumocystisorganisms found in formalin-fixed paraffin-embedded (FFPE) lung tissue sections from Chilean sudden infant death (SID) victims. This approach needed first to optimize a DNA extraction method from such histological sections. For that purpose, the QIAamp DNA Isolation from Paraffin-Embedded Tissue method (Qiagen) was first tested on FFPE lung tissue sections of immunosuppressed Wistar rats inoculated with rat-derivedPneumocystis. Successful DNA extraction was assessed by the amplification of a 346 bp fragment of the mitochondrial large subunit rRNA gene of thePneumocystisspecies using a previously described PCR assay. PCR products were analysed by direct sequencing and sequences corresponding toPneumocystis cariniiwere found in all the samples. This method was then applied to FFPE lung tissue sections from Chilean SID victims.Pneumocystis jiroveciiwas successfully identified in the three tested samples. In conclusion, an efficient protocol for isolating PCR-ready DNA from FFPE lung tissue sections was developed. It established that thePneumocystisspecies found in the lungs of Chilean SID victims wasP. jirovecii.
Amino sugar phosphate levels in Giardia change during cyst wall formation
Authors: Sener Keriman , Shen Zuojun , Newburg David S. , Jarroll Edward L.
Published: May 2004

The parasite Giardia intestinalis exists as a trophozoite (vegetative) that infects the human small intestine, and a cyst (infective) that is shed in host faeces. Cyst viability in the environment depends upon a protective cyst wall, which consists of proteins and a unique β(1-3) GalNAc homopolymer. UDP-GalNAc, the precursor for this polysaccharide, is synthesized from glucose by an enzyme pathway that involves amino sugar phosphate intermediates. Using a novel method of microanalysis by capillary electrophoresis, the levels of amino sugar phosphate intermediates in trophozoites before encystment, during a period of active encystment and after the peak of encystment were measured. These levels were used to deduce metabolic control of amino sugar phosphates associated with encystment. Levels of amino sugar phosphate intermediates increased during encystment, and then decreased to nearly non-encysting levels. The most pronounced increase was in glucosamine 6-phosphate, which is the first substrate unique in this pathway, and which is the positive effector for the pathway's putative rate-controlling enzyme, UDP-GlcNAc pyrophosphorylase. Moreover, more UDP-GalNAc than UDP-GlcNAc, its direct precursor, was detected at 24 h. It is postulated that the enhanced UDP-GalNAc is a result of enhanced synthesis of UDP-GlcNAc by the pyrophosphorylase, and its preferential conversion to UDP-GalNAc. These results suggest that kinetics of amino sugar phosphate synthesis in encysting Giardia favours the direction that supports cyst wall synthesis. The enzymes involved in synthesis of UDP-GalNAc and its conversion to cyst wall might be potential targets for therapeutic inhibitors of Giardia infection.
PCR-based identification of zoonotic isolates of Blastocystis from mammals and birds
Authors: Yoshikawa Hisao , Abe Niichiro , Wu Zhiliang
Published: May 2004

The genotype of Blastocystis isolated from humans and animals is highly polymorphic. Therefore, it is important to compare the genotypes of Blastocystis isolates from humans and animals to determine the zoonotic potential of animal isolates. PCR-based genotype classification using known sequence-tagged site (STS) primers allows identification of zoonotic isolates of animal origin. To this end, 51 isolates from monkeys, cattle, pigs, chickens, quails and pheasants were subjected to genotype analysis using seven kinds of STS primers. Out of the 51 isolates, 39 were identified as one of the known genotypes, four showed mixed genotypes, and eight were unknown genotypes as these were negative for all STS primers. When these results were combined with previous studies on 41 isolates from animals and compared with the diversity of genotypes of 102 human Blastocystis hominis isolates, 67·4 % (62/92) of isolates from mammals and birds were identical to human B. hominis genotypes. Since the unknown genotype of human origin had been placed into an additional clade in the small-subunit rRNA gene phylogeny, further molecular study on the eight isolates of unknown genotype from the present study will facilitate our understanding of their zoonotic potential.
tvcp12: a novel Trichomonas vaginalis cathepsin L-like cysteine proteinase-encoding gene
Authors: León-Sicairos Claudia R. , León-Félix Josefina , Arroyo Rossana
Published: May 2004

Trichomonas vaginalis is the causative agent of trichomoniasis, one of the most common sexually transmitted diseases in humans. This protozoan has multiple proteinases that are mainly of the cysteine proteinase (CP) type, some of which are known to be involved in the parasite's virulence. Here, a novel T. vaginalis CP-encoding gene, tvcp12, was identified and characterized. tvcp12 is 948 bp long and encodes a predicted 34·4 kDa protein that has the characteristics of the papain-like CP family. TvCP12 does not appear to have a signal peptide, suggesting that this is a cytoplasmic CP. By Southern blot assays, the tvcp12 gene was found as a single copy in the T. vaginalis genome. Remarkably, Northern blot experiments showed a single transcript band of ∼1·3 kb in the mRNA obtained from parasites grown in low iron conditions and no transcript was observed in the mRNA from parasites grown in high iron conditions. By RT-PCR assays, a 270 bp band was amplified from the cDNA of parasites grown in low iron medium, which was very faint when cDNA from parasites grown in high iron conditions was used. Transcripts of the 3′ region obtained in both iron conditions presented differences in their poly(A) tail length. These data suggest that tvcp12 is another gene that is negatively regulated by iron and that the length of the poly(A) tail may be one of the factors involved in the iron-modulated protein expression.
Production of ammonia by Tritrichomonas foetus and Trichomonas vaginalis
Authors: Kleydman Yekaterina , Yarlett Nigel , Gorrell Thomas E.
Published: May 2004

Production of ammonia is difficult to find among the various studies of amino acid metabolism in protozoa. Several studies suggest that catabolism of arginine to ammonium is important for the growth of trichomonads. Trichomonads are amitochondriate zooflagellates that thrive under microaerophilic and anaerobic conditions. The authors were able to detect accumulation of ammonium ions and ammonia in cultures of Tritrichomonas foetus and Trichomonas vaginalis, including those resistant to metronidazole. Ammonium ions and ammonia were detected using the indophenol colorimetric method. Cells incubated overnight under an ambient oxygen gas phase had 0·9 mM soluble ammonium (NH4
+ and NH3) or a 20 % greater concentration of ammonium relative to sterile growth medium that had been incubated similarly. Production of ammonia itself was confirmed by analysis of a wick that was moistened with sulfuric acid (20 mM) and placed above the liquid in sealed cultures of a strain of Trichomonas vaginalis. The wicks from these cultures captured the equivalent of 0·048 mM volatile ammonia (NH3) from the liquid as compared to 0·021 mM volatile ammonia from sterile medium after overnight incubation. Intact trichomonads, 0·7×106 cells ml−1 equivalent to 0·7 mg protein ml−1, incubated in Doran's buffer with or without (1 mM) l-arginine produced significant amounts of soluble ammonium (0·07 mM and 0·04 mM, respectively) during 60 min. The results indicate that ammonium ions and the more irritating ammonia are significant metabolites of trichomonads. In addition, based upon end-product amounts, it appears that the rate of arginine metabolism is of the same order of magnitude as that for carbohydrate metabolism by trichomonads.
The last step in coenzyme B12 synthesis is localized to the cell membrane in bacteria and archaea
Authors: Maggio-Hall Lori A. , Claas Kathy R. , Escalante-Semerena Jorge C.
Published: May 2004

InSalmonella enterica, the last step of the synthesis of adenosylcobamide is catalysed by the cobalamin synthase enzyme encoded by thecobSgene of this bacterium. Overexpression of theS. enterica cobSgene inEscherichia colielicited the accumulation of the phage shock protein PspA, a protein whose expression has been linked to membrane stress. Resolution of inner and outer membranes ofS. entericaby isopycnic density ultracentrifugation showed CobS activity associated with the inner membrane, a result that was confirmed using antibodies against CobS. Computer analysis of the predicted amino acid sequence of CobS suggested it was an integral membrane protein. Results of experiments performed with strains carrying plasmids encoding CobS–alkaline phosphatase or CobS–β-galactosidase protein fusions were consistent with the membrane localization of the CobS protein. Modifications to the predicted model were made based on data obtained from experiments using protein fusions. The function encoded by thecobSorthologue in the methanogenic archaeonMethanobacterium thermoautotrophicumstrain ΔH compensated for the lack of CobS during cobalamin synthesis incobSstrains ofS. enterica. Cobalamin synthase activity was also detected in a membrane preparation ofM. thermoautotrophicum. It was concluded that the assembly of the nucleotide loop of adenosylcobamides in archaea and bacteria is a membrane-associated process. Possible reasons for the association of adenosylcobamide biosynthetic enzymes with the cell membrane are discussed.
Increased transcription rates correlate with increased reversion rates in leuB and argH Escherichia coli auxotrophs
Authors: Reimers Jacqueline M. , Schmidt Karen H. , Longacre Angelika , Reschke Dennis K. , Wright Barbara E.
Published: May 2004

Escherichia coliauxotrophs ofleuBandargHwere examined to determine if higher rates of transcription in derepressed genes were correlated with increased reversion rates. Rates ofleuBandargHmRNA synthesis were determined using half-lives and concentrations, during exponential growth and at several time points during 30 min of amino acid starvation. Changes in mRNA concentration were primarily due to increased mRNA synthesis and not to increased stability. Four strains ofE. coliamino acid auxotrophs, isogenic except forrelAandargR, were examined. In both theleuBandargHgenes, rates of transcription and mutation were compared. In general, strains able to activate transcription with guanosine tetraphosphate (ppGpp) had higher rates of mRNA synthesis and mutation than those lacking ppGpp (relA2mutants).argRknockout strains were constructed inrelA+andrelAmutant strains, and rates of bothargHreversion and mRNA synthesis were significantly higher in theargRknockouts than in the regulated strains. A statistically significant linear correlation between increased rates of transcription and mutation was found for data from both genes. In general, changes in mRNA half-lives were less than threefold, whereas changes in rates of mRNA synthesis were often two orders of magnitude. The results suggest that specific starvation conditions target the biosynthetic genes for derepression and increased rates of transcription and mutation.
The Fur-like protein Mur of Rhizobium leguminosarum is a Mn2+-responsive transcriptional regulator
Authors: Díaz-Mireles E. , Wexler M. , Sawers G. , Bellini D. , Todd J. D. , Johnston A. W. B.
Published: May 2004

In wild-type Rhizobium leguminosarum, the sitABCD operon specifies a Mn2+ transporter whose expression is severely reduced in cells grown in the presence of this metal. Mutations in the R. leguminosarum gene, mur (manganese uptake regulator), whose product resembles the Fur transcriptional regulator, cause high-level expression of sitABCD in the presence of Mn2+. In gel-shift mobility assays, purified R. leguminosarum Mur protein bound to at least two regions near the sitABCD promoter region, although this DNA has no conventional consensus Fur-binding sequences (fur boxes). Thus, in contrast to γ-proteobacteria, where Fur binds Fe2+, the R. leguminosarum Fur homologue, Mur, act as a Mn2-responsive transcriptional regulator.
Molecular and functional characterization of type I signal peptidase from Legionella pneumophila
Authors: Lammertyn Elke , Van Mellaert Lieve , Meyen Eef , Lebeau Ilya , De Buck Emmy , Anné Jozef , Geukens Nick
Published: May 2004

Legionella pneumophilais a facultative intracellular Gram-negative rod-shaped bacterium that has become an important cause of both community-acquired and nosocomial pneumonia. Numerous studies concerning the unravelling of the virulence mechanism of this important pathogen have been initiated. As evidence is now accumulating for the involvement of protein secretion systems in bacterial virulence in general, the type I signal peptidase (LepB) ofL. pneumophilawas of particular interest. This endopeptidase plays an essential role in the processing of preproteins carrying a typical amino-terminal signal peptide, upon translocation across the cytoplasmic membrane. This paper reports the cloning and the transcriptional analysis of theL. pneumophila lepBgene encoding the type I signal peptidase (SPase). Reverse transcription PCR experiments showed clearlepBexpression whenL. pneumophilawas grown both in culture medium, and also intracellularly inAcanthamoeba castellanii, a natural eukaryotic host ofL. pneumophila. In addition, LepB was shown to be encoded by a polycistronic mRNA transcript together with two other proteins, i.e. a LepA homologue and a ribonuclease III homologue. SPase activity of the LepB protein was demonstrated byin vivocomplementation analysis in a temperature-sensitiveEscherichia coli lepBmutant. Protein sequence and predicted membrane topology were compared to those of leader peptidases of other Gram-negative human pathogens. Most strikingly, a strictly conserved methionine residue in the substrate binding pocket was replaced by a leucine residue, which might influence substrate recognition. Finally it was shown byin vivoexperiments thatL. pneumophilaLepB is a target for (5S,6S)-6-[(R)-acetoxyethyl]-penem-3-carboxylate, a specific inhibitor of type I SPases.