bluuebunny/crossref_metadata_2025 huggingface dataset:
Assembly proteins of CS1 pili of enterotoxigenic Escherichia coli
Authors: Sakellaris Harry , Balding Donna P. , Scott June R.
Published: October 2006

Some strains of enterotoxigenic Escherichia coli associated with human diarrhoeal disease produce a class of pili represented by those called CS1. For the assembly of the major‐pilin subunit, CooA, into pili, each of four linked genes, cooB,A,C, and D, is required. In this study, we have determined the subcellular localization of CooB, C and D, and investigated the molecular interactions of these proteins using specific antisera. CooD appears to be an integral pilus protein because it co‐purifies with, and is strongly associated with, CS1 pili. In keeping with its role as an assembly protein, the CooD minor pilin (when overexpressed in CS1‐piliated strains) was detected in periplasmic inter‐molecular complexes with the major‐pilin subunit CooA. CooB is an assembly protein found exclusively in the periplasm of CS1‐piliated strains. CooB also forms periplasmic intermolecular complexes with CooA, but does not constitute part of the final pilus structure. Immunoblot analysis of cell fractions showed that CooC is an outer membrane protein of CS1‐piliated E. coli. Based on this information, we have proposed a model for CS1 ‐pilus assembly which is very similar to the model for polymerization of the PapA pilin of uropathogenic E. coli. As the assembly proteins of Pap and CS1 pili are structurally unrelated, this may represent a case of convergent evolution.
Internalin must be on the bacterial surface to mediate entry of Listeria monocytogenes into epithelial cells
Authors: Lebrun Maryse , Mengaud Jérôme , Ohayon Hélène , Nato Farida , Cossart Pascale
Published: October 2006

Entry of Listeria monocytogenes into cultured epithelial cells requires production of internalin, a protein with features characteristic of some Gram‐positive bacterial surface proteins, in particular an LPXTG motif preceding a hydrophobic sequence and a few basic residues at its C‐terminal end. By immunofluorescence and immunogold labelling, we show that in wild‐type L. monocytogenes, internalin is present on the cell surface and has a polarized distribution similar to that of ActA, another surface protein of L. monocytogenes involved in actin assembly. Through a genetic analysis, we establish that the C‐terminal region of internalin is necessary for cell‐surface association, and that although internalin is partially released in the culture medium, its location on the bacterial surface is required to promote entry. Finally, using a‘domain‐swapping’strategy ‐ replacement of the cell wall anchor of InIA by the membrane anchor of ActA ‐ we show that the reduced ability to adhere and enter cells of strains expressing InIA‐ActA correlates with a lower amount of surface‐exposed internalin. Taken together, these results suggest that internalin exposed on the bacterial surface mediates direct contact between the bacterium and the host cell.
Activation of transcription at divergent urea‐dependent promoters by the urease gene regulator UreR
Authors: D'Orazio Sarah E. F. , Thomas Venetta , Collins Carleen M.
Published: October 2006

The Proteus mirabilis and plasmid‐encoded urease loci contain seven contiguous structural and accessory genes (ureDABCEFG) and the divergently transcribed ureR, which codes for an AraC‐like transcriptional activator. Previously, it was shown that the plasmid‐encoded ureR to ureD intergenic region contained divergent promoters (ureRp and ureDp). Transcription from these promoters required both the effector molecule urea and the activator protein UreR. In this report, we demonstrate that the P. mirabilis urease gene cluster contains similar divergent urea‐ and UreR‐dependent promoters. The ureR gene products from either urease locus were able to activate transcription at both the plasmid‐encoded and P. mirabilis promoters. The minimal concentration of urea required to activate transcription at ureRp or ureDp from either gene cluster was approximately 4 mM. The transcriptional start sites for the plasmid‐encoded and P. mirabilis divergent promoters were similar in an Escherichia coli DH5α background, as determined by primer‐extension analysis. However, in P. mirabilis HI4320, transcription of ureR initiated predominately at an alternative site. Physical mapping and inhibition studies were used to localize the UreR‐binding sites within the plasmid‐encoded ureRp and ureDp intergenic sequences to regions of 68 bp and 86 bp, respectively. Gel shift analysis demonstrated that UreR bound to a 135 bp fragment in the approximate centre of the plasmid‐encoded ureR to ureD intergenic region. The results presented here suggest that the P. mirabilis and plasmid‐encoded urease gene clusters utilize similar mechanisms of transcriptional activation in response to urea.
Customized secretion chaperones in pathogenic bacteria
Authors: Wattiau Pierre , Woestyn Sophie , Cornelis Guy R.
Published: October 2006

Pathogenic yersiniae secrete about a dozen anti‐host proteins, the Yops, by a pathway which does not involve cleavage of a classical signal peptide. The Yop secretory apparatus, called Ysc, for Yop secretion, is the archetype of type III secretion systems (which serve for the secretion of virulence proteins by several animal and plant pathogens) and is related to the flagellar assembly apparatus. The Yop secretion signal is N‐terminal but has not been defined to date. Apart from the Ysc machinery, secretion of at least four Yops requires cytoplasmic proteins called Syc (for specific Yop chaperone). Each Syc protein binds to its cognate Yop. Unlike most cytoplasmic chaperones, these proteins do not have an ATP‐binding domain, and are presumably devoid of ATPase activity. They share a few common properties: an acidic pl, a size in the range of 15–20 kDa, and a putative amphipathic α‐helix in the C‐terminal portion. They were recently shown to have counterparts in other pathogenic bacteria, where they appear to have a similar function.
Optimized BlaM‐transposon shuttle mutagenesis of Helicobacter pylori allows the identification of novel genetic loci involved in bacterial virulence
Authors: Odenbreit Stefan , Till Markus , Haas Rainer
Published: October 2006

Helicobacter pylori is an important etiologic agent of gastroduodenal disease in humans. In this report, we describe a general genetic approach for the identification of genes encoding exported proteins in H. pylori. The novel TnMax9 mini‐blaM transposon was used for insertion mutagenesis of a H. pylori gene library established in Escherichia coli. A total of 192 E. coli clones expressing active β‐lactamase fusion proteins (BlaM+) were obtained, indicating that the corresponding target plasmids carry H. pylori genes encoding putative extracytoplasmic proteins. Natural transformation of H. pylori P1 or P12 using the 192 mutant plasmids resulted in 135 distinct H. pylori mutant strains (70%). Screening of the H. pylori collection of mutant strains allowed the identification of mutant strains impaired in motility, in natural transformation competence and in adherence to gastric epithelial cell lines. Motility mutants could be grouped into distinct classes: (i) mutant strains lacking the major flagellin subunit FlaA and intact flagella (class I); (ii) mutant strains with apparently normal flagella, but reduced motility (class II), and (iii) mutant strains with obviously normal flagella, but completely abolished motility (class III). Two independent mutations that exhibited defects in natural competence for genetic transformation mapped to different genetic loci. In addition, two independent mutant strains were isolated by their failure to bind to the human gastric carcinoma cell line Katoill. Both mutant strains carried a transposon in the same gene, 0.8 kb apart, and showed decreased autoagglutination when compared to the wild‐type strain.
Purification and functional characterization of PecS, a regulator of virulence‐factor synthesis in Erwinia chrysanthemi
Authors: Praillet Thierry , Nasser William , Robert‐Baudouy Janine , Reverchon Sylvie
Published: October 2006

The Erwinia chrysanthemi pecS gene encodes a repressor that negatively regulates the expression of virulence factors such as pectinases or cellulases. The cloned pecS gene was overexpressed using a phage T7 system. The purification of PecS involved DEAE‐anion exchange and TSK‐heparin columns and delivered the PecS protein that was purified to homogeneity. The purified repressor displayed an 18 kDa apparent molecular mass and an isoelectric point near to neutrality (PI = 6.5). Gel‐filtration experiments revealed that the PecS protein is a dimer. Bandshift assays demonstrated that the PecS protein could specifically bind in vitro to the regulatory sites of the in vivo PecS‐regulated genes. The interaction between the PecS protein and its DNA‐binding site was characterized by a relatively low affinity (about 10−8 M). DNase I footprintings revealed short protected sequences only with the most in vivo PecS‐regulated genes. Alignment of these PecS‐binding sites did not show a well‐conserved consensus sequence. lmmunoblotting demonstrated that the copy number of the PecS protein was approximately 50 dimers per cell. The low affinity of the PecS repressor for its DNA targets and the low cellular PecS content suggest the existence of E. chrysanthemi‐specific factors able to potentiate PecS protein activity in vivo.
Reciprocal regulation of the differentiation of Myxococcus xanthus by Pkn5 and Pkn6, eukaryotic‐like Ser/Thr protein kinases
Authors: Zhang Wandong , Lnouye Masayori , Inouye Sumiko
Published: October 2006

Myxococcus xanthus contains a large family of genes encoding eukaryotic‐like serinehhreonine kinases. Among them, two genes, pkn5 and pkn6, are divergently located on the chromosome and share a 46 bp promoter region between their transcription initiation sites, as determined by RNA protection. Pkn5, consisting of 380 amino acid residues, is a soluble protein in the cytoplasm, while Pkn6, consisting of 710 amino acid residues, is a transmembrane protein. Its membrane topology was determined using the Pkn6‐PhoA fusion protein in Escherichia coli, which has a single transmembrane domain with the N‐terminal domain in the cytoplasm and the C‐terminal domain outside the cytoplasmic membrane. Both proteins, when expressed in E. coli, were autophosphorylated: Pkn5 only at Ser, and Pkn6 at both Ser and Thr. In M. xanthus, both genes are expressed constitutively throughout the life cycle, with slight increases at an early stage of development. Most strikingly, a pkn5‐deletion strain forms fruiting bodies much faster than the wild‐type strain, while a pknb‐deletion strain develops slower than the wild‐type strain. These results, together with the fact that the pkn5‐deletion strain is able to form fruiting bodies on semi‐rich media, suggest that Pkn5 and Pkn6 have reciprocal roles in M. xanthus growth and development. Furthermore, Pkn6 may be a transmembrane sensor of external signals for development, while Pkn5 is a kinase that negatively regulates M. xanthus development.
Construction of a ferritin‐deficient mutant of Campylobacter jejuni: contribution of ferritin to iron storage and protection against oxidative stress
Authors: Wai Sun Nyunt , Nakayama Koji , Umene Kenichi , Moriya Tetsuhiro , Amako Kazunobu
Published: October 2006

The ferritin‐encoding gene (cft) of Campylobacter jejuni was cloned and sequenced. The nucleotide sequence of cft had a 501 bp open reading frame for a protein with 167 amino acids and a predicted molecular mass of 19180 Da, and showed a high similarity to that of Helicobacter pylori and Escherichia coli ferritin genes. To determine the biological function of ferritin in C. jejuni, a ferritin‐deficient mutant was constructed. The growth of ferritin‐deficient strain SNA1 was clearly inhibited under iron deprivation. The ferritin‐deficient mutant was more sensitive to killing by H2O2 and paraquat than the isogenic parent strain. These findings demonstrate that ferritin in C. jejuni makes a significant contribution to both iron storage and protection from intracellular iron overload, and resulting iron‐mediated oxidative stress.
Role of recombinational repair in sensitivity to an antitumour agent that inhibits bacteriophage T4 type II DNA topoisomerase
Authors: Neece Sue H. , Carles‐Kinch Kelly , Tomso Daniel J. , Kreuzer Kenneth N.
Published: October 2006

The bacteriophage T4‐encoded type II DNA topoisomerase is the major target for the antitumour agent m‐AMSA (4‐(9‐acridinylamino)methanesulphon‐m‐anisidide) in phage‐infected bacterial cells. Inhibition of the purified enzyme by m‐AMSA results in formation of a cleavage complex that contains the enzyme covalently attached to DNA on both sides of a double‐strand break. In this article, we provide evidence that this cleavage complex is responsible for inhibition of phage growth and that recombinational repair can reduce sensitivity to the antitumour agent, presumably by eliminating the complex (or some derivative thereof). First, topoisomerase‐deficient mutants were shown to be resistant to m‐AMSA, indicating that m‐AMSA inhibits growth by inducing the cleavage complex rather than by inhibiting enzyme activity. Second, mutations in several phage genes that encode recombination proteins (uvsX, uvsY, 46 and 59) increased the sensitivity of phage T4 to m‐AMSA, strongly suggesting that recombination participates in the repair of topoisomerase‐mediated damage. Third, m‐AMSA stimulated recombination in phage‐infected bacterial cells, as would be expected from the recombinational repair of DNA damage. Finally, m‐AMSA induced the production of cleavage complexes involving the T4 topoisomerase within phage‐infected cells.
The activation of PhoB by acetylphosphate
Authors: McCleary W. R.
Published: October 2006

PhoB is a response‐regulator protein from Escherichia coli that controls an adaptive response to limiting phosphate. It is activated by autophosphorylation of a conserved aspartate residue within its regulatory domain. Its primary phospho‐donor is its cognate histidine kinase PhoR; however, it also becomes phos‐phorylated when incubated with acetylphosphate. To further characterize its activation, PhoB was considered to be an acetylphosphatase whose enzymatic mechanism involves a phospho‐enzyme intermediate. The kinetic constants for autophosphorylation were determined using 32P‐ and fluorescence‐based assays and indicated that PhoB has a Km for acetylphosphate of between 7 and 8 mM. These constants are not consistent with an in vivo role for acetylphosphate in the normal control of the Pho regulon. In addition, when PhoB was phosphorylated by acetylphosphate it eluted from a high‐performance liquid chromatography (HPLC) size‐exclusion column in two peaks. The larger form of PhoB eluted from the column in a similar manner to a chemically cross‐linked dimer of PhoB. The smaller form of PhoB is a monomer. Phosphorylated PhoB bound pho‐box DNA approximately 10 times tighter than PhoB. These observations show that PhoB forms a dimer when phosphorylated and suggest that the characteristics of activated PhoB result from its dimer ization.
The F plasmid traY gene product binds DNA as a monomer or a dimer: structural and functional implications
Authors: Nelson William C. , Matson Steven W.
Published: October 2006

The F factor traY gene product (TraYp) is a site‐specific DNA‐binding protein involved in initiation of DNA transfer during bacterial conjugation. The sequence of TraYp exhibits a unique direct‐repeat structure predicted to have a ribbon‐helix‐helix DNA‐binding motif in each repeat unit. The stoichiometry of TraYp binding to DNA was determined to further support the hypothesis that TraYp is a member of the ribbon‐helix‐helix family of DNA‐binding proteins. A gluta‐thione‐S‐transferase‐traY fusion protein was purified and shown to possess almost wild‐type DNA‐binding activity. DNA‐binding experiments were performed in which the DNA ligand was incubated with either the fusion protein, the wild‐type protein, or both. The results indicate that TraYp can bind DNA as a monomer or a dimer. Thus a TraYp monomer folds into a stable three‐dimensional structure similar to that of a dimer of the ribbon‐helix‐helix proteins Arc or Mnt. A homology model of a TraYp monomer has been constructed using the co‐crystal structure of Arc bound to DNA as a template to provide additional support for this conclusion. In addition, we have shown that an origin of the transfer‐deletion mutant lacking approximately half of the TraYp‐binding site can only be bound by a monomer of TraYp. The functional implications of this result are discussed.
Mutational analysis and properties of the msbA gene of Escherichia coli, coding for an essential ABC family transporter
Authors: Polissi Alessandra , Georgopoulos Costa
Published: October 2006

The htrB gene was discovered because its insertional inactivation interfered with Escherichia coli growth and viability at temperatures above 32.5°C, as a result of accumulation of phospholipids. The msbA gene was originally discovered because when cloned on a low‐copy‐number plasmid vector it was able to suppress the temperature‐sensitive growth phenotype of an htrB null mutant as well as the accumulation of phospholipids. The msbA gene product belongs to the superfamily of ABC transporters, a universally conserved family of proteins characterized by a highly conserved ATP‐binding domain. The msbA gene is essential for bacterial viability at all temperatures. In order to understand the physiological role of the MsbA protein, we mutated the ATP‐binding domain using random PCR mutagenesis. Six independent mutants were isolated and characterized. Four of these mutations resulted in single‐amino‐acid substitutions in non‐conserved residues and were able to support cell growth at 30°C but not at 43°C. The remaining two mutations behaved as recessive lethals, and resulted in single‐amino‐acid substitutions in Walker motif B, one of the two highly conserved regions of the ATP‐binding domain. Despite the fact that neither of these two mutant proteins can support E. coli growth, they both retained the ability to bind ATP in vitro. In addition, we present evidence to show that W‐acetyl [3H]‐glucosamine, a precursor of lipopolysaccharides, accumulates at the non‐permissive temperature in the inner membrane of either htrB null or msbA conditional lethal strains. Translocation of the precursor to the outer membrane is restored by transformation with a plasmid containing the wild‐type msbA gene. A possible role for MsbA
The cytosolic SycE and SycH chaperones of Yersinia protect the region of YopE and YopH involved in translocation across eukaryotic cell membranes
Authors: Woestyn Sophie , Sory Marie‐Paule , Boland Anne , Lequenne Olivier , Cornelis Guy R.
Published: October 2006

Yersinia adhering at the surface of eukaryotic cells secrete a set of proteins called Yops. This secretion which occurs via a type III secretion pathway is immediately followed by the injection of some Yops into the cytosol of eukaryotic cells. Translocation of YopE and YopH across the eukaryotic cell membranes requires the presence of the translocators YopB and YopD. YopE and YopH are modular proteins composed of an N‐terminal secretion signal, an internalization domain, and an effector domain. Secretion of YopE and YopH requires the presence of the specific cytosolic chaperones SycE and SycH, respectively. In this work, we have mapped the regions of YopE and YopH that are involved in binding of their cognate chaperone. There is only one Syc‐binding domain in YopE (residues 15–50) and YopH (residues 20–70). This domain is localized immediately after the secretion signal and it corresponds to the internalization domain. Removal of this bifunctional domain did not affect secretion of YopE and YopH and even suppressed the need for the chaperone in the secretion process. Thus SycE and SycH are not secretion pilots. Instead, we propose that they prevent intrabacterial interaction of YopE and YopH with proteins involved in translocation of these Yops across eukaryotic cell membranes.
A novel DnaJ‐like protein in Escherichia coli inserts into the cytoplasmic membrane with a Type III topology
Authors: Clarke David J. , Jacq A. , Holland I. B.
Published: October 2006

We describe a novel Escherichia coli protein, DjlA, containing a highly conserved J‐region motif, which is present in the DnaJ protein chaperone family and required for interaction with DnaK. Remarkably, DjlA is shown to be a membrane protein, localized to the inner membrane with the unusual Type III topology (N‐out, C‐in). Thus, DjlA appears to present an extremely short N‐terminus to the periplasm and has a single transmembrane domain (TMD) and a large cytoplasmic domain containing the C‐terminal J‐region. Analysis of the TMD of DjIA and recently identified homologues in Coxiella burnetti and Haemophilus influenzae revealed a striking pattern of conserved glycines (or rarely alanine), with a four‐residue spacing. This motif, predicted to form a spiral groove in the TMD, is more marked than a repeating glycine motif, implicated in the dimerization of TMDs of some eukaryotic proteins. This feature of DjlA could represent a promiscuous docking mechanism for interaction with a variety of membrane proteins. DjlA null mutants can be isolated but these appear rapidly to accumulate suppressors to correct envelope and growth defects. Moderate (10‐fold) overproduction of DjlA suppresses a mutation in FtsZ but markedly perturbs cell division and cell‐envelope growth in minimal medium. We propose that DjlA plays a role in the correct assembly, activity and/or maintenance of a number of membrane proteins, including two‐component signal‐transduction systems.
Developmentally regulated synthesis of an unusually small, basic peptide by Coxiella burnetii
Authors: Heinzen R. A. , Howe D. , Mallavia L. P. , Rockey D. D. , Hackstadt T.
Published: October 2006

Coxiella burnetii undergoes a poorly defined developmental cycle within phagolysosomes of eukaryotic host cells. Two distinct developmental forms are part of this cycle: a small‐cell variant (SCV) and large‐cell variant (LCV). Ultrastructurally, the SCV is distinguished from the LCV by its smaller size and condensed chromatin. At a molecular level, little is known about morphogenesis in C. burnetii, and no proteins specific to the SCV have been identified. Preparative isoelectric focusing was conducted to purify basic proteins possibly involved in SCV chromatin structure. A predominant protein of low Mr was present in the most basic fraction, eluting with a pH of approx. 11. Degenerate deoxyoligonucleotides corresponding to the N‐terminal sequence of this protein were used to recover a cosmid clone from a C. burnetii genomic library. Nucleotide sequencing of insert DNA revealed an open reading frame designated scvA (small‐cell‐variant protein A) with coding potential for a 30 amino acid protein (ScvA) with a predicted Mr of 3610. ScvA is 46% arginine plus 46% glutamine with a predicted pi of 12.6. SDS‐PAGE and silver staining of lysates of SCV and LCV purified by caesium chloride‐equilibrium density centrifugation revealed a number of proteins unique to each cell type. Immunoblot analysis with ScvA antiserum demonstrated the presence of ScvA only in the SCV. By immunoelectron microscopy, ScvA antiserum labelled only the SCV, with the label concentrated on the condensed nucleoid. In addition, ScvA bound double‐stranded DNA in gel mobility‐shift assays. A 66% reduction in the mean number of gold particles per Coxiella cell was observed at 12 h post‐infection when compared with the starting inoculum. Collectively, these data suggest that synthesis of ScvA is developmentally regulated, and that the protein may serve a structural or functional role as an integral component of the SCV chromatin. Moreover, degradation of this protein may be a necessary prerequisite for morphogenesis from SCV to LCV.
An elongation factor Tu (EF‐Tu) resistant to the EF‐Tu inhibitor GE2270 in the producing organism Planobispora rosea
Authors: Sosio Margherita , Amati Giuseppe , Cappellano Carmela , Sarubbi Edoardo , Monti Federica , Donadio Stefano
Published: October 2006

Using a cell‐free protein‐synthesis system, we have established that the elongation factor (EF) Tu (EF‐Tu) of the actinomycete Planobispora rosea, the producer of the thiazolyl peptide GE2270, a specific EF‐Tu inhibitor, is highly resistant to its own antibiotic, while it is completely inhibited by kirromycin, which is another inhibitor of this factor. P. rosea was found to possess a single tuf gene, located between fus and rpsJ, encoding other components of the protein‐synthesis machinery. The P. rosea tuf gene was expressed as a translations! fusion to maIE in Escherichia coli, and the resulting EF‐Tu with an N‐terminal Gly‐Met extension was able to promote poly(U)‐directed poly(Phe) synthesis in cell‐free systems. This activity was not affected by GE2270, and the recombinant protein was incapable of binding the antibiotic, indicating that the P. rosea EF‐Tu is intrinsically resistant to this inhibitor. Inspection of the translated tuf sequence revealed a number of amino acid substitutions in highly conserved positions. These residues, which are likely to be involved in conferring GE2270 resistance, map in EF‐Tu domain II, as do the only two known mutations conferring resistance to this class of thiazolyl peptides in Bacillus subtilis.
Regulated expression of the dinR and recA genes during competence development and SOS induction in Bacillus subtilis
Authors: Haijema Bert Jan , van Sinderen Douwe , Winterling Kevin , Kooistra Jan , Venema Gerard , Hamoen Leendert Willem
Published: October 2006

It has been hypothesized that the dinR gene product of Bacillus subtilis acts as a repressor of the SOS regulon by binding to DNA sequences located upstream of SOS genes, including dinR and recA. Following activation as a result of DNA damage, RecA is believed to catalyse DinR‐autocleavage, thus derepressing the SOS regulon. The present results support this hypothesis: a dinR insertion mutation caused a high, constitutive expression of both dinR and recA, which could not be further elevated by SOS‐induction. In addition, gel‐retardation assays demonstrated a direct interaction between the dinR gene product and the recA and dinR promoter regions. Epistatic interactions and gel‐retardation assays demonstrated that the previously reported competence‐specific expression of recA directly depended upon the gene product of comK, the competence transcription factor. These data demonstrate the existence of a direct regulatory link between the competence signal‐transduction pathway and the SOS regulon.
Modelling temperature‐dependent bionomics of Bemisia tabaci (Q‐biotype)
Authors: BONATO OLIVIER , LURETTE AMANDINE , VIDAL CLAIRE , FARGUES JACQUES
Published: October 2006

Abstract The influence of temperature (17, 21, 25, 30 and 35 °C) on life‐history traits of a Q‐biotype Bemisia tabaci population on tomato is studied. Temperature‐dependent relationships are characterized for immature developmental rate, immature survival, fecundity, longevity and intrinsic rate of increase. Development time vary from 20 days at 30 °C to 56 days at 17 °C and the lowest thermal threshold is estimated at 10.2 °C. The optimal temperature for immature development is 32.5 °C. Total fecundity (eggs per female) ranges from 105.3 (at 21 °C) to 41 (at 35 °C). The longevity decreases with temperature increase. The intrinsic rate of increase ranges from 0.0450 (at 17 °C) to 0.123 (at 30 °C). The functional relationships between temperature and life‐history parameters are used to evaluate the effect of temperature on the population dynamics. Such mathematical relationships could provide a basis for future development of population models.
Preparation, Characterization and Epitope Mapping of Monoclonal Antibodies Specific to Human Mast Cell Carboxypeptidase
Authors: Zhou Y. , Chen Z. , He S.
Published: October 2006

Human mast cell carboxypeptidase (hMC‐CP) is a unique product of mast cells. Unlike tryptase and chymase, its potential function and expression in diseased conditions remain largely unknown. To develop an assay for hMC‐CP, the recombinant fusion protein of hMC‐CP and purified native skin hMC‐CP was prepared, and two novel monoclonal antibodies against hMC‐CP named CCP1 (IgG1 isotype) and CCP2 (IgM isotype) were raised in the present study. Epitope analysis shows that CCP1 and CCP2 antibodies recognize epitopes located in the region of amino acids 112–202 of hMC‐CP, and hydrophilicity analysis implies that epitopes might be located in the amino acid residues 123–134 and 165–177. Furthermore, using a competition enzyme‐linked immunosorbent assay, it was shown that the epitope recognized by CCP1 is close to that recognized by CCP2 or the two antibodies partially share the same epitope. Flow cytometry analysis shows that basophilic leukemia cell line KU812 reacts with both CCP1 and CCP2 antibodies, suggesting that this cell line expresses hMC‐CP. In conclusion, although the two antibodies possess different isotypes, they may partially share the same epitope. These two antibodies will be valuable tools for the development of an assay to detect the levels of hMC‐CP in the biological fluids in man.
B‐cell CD25 Expression in Murine Primary and Secondary Lymphoid Tissue
Authors: Amu S. , Gjertsson I. , Tarkowski A. , Brisslert M.
Published: October 2006

B cells are in analogy with T cells capable of expressing functional IL‐2 receptors. IL‐2R α‐chain (CD25) positive T cells have been studied in detail but not much is known about CD25 positive B cells. The aim of this study was to examine the phenotypic properties of the CD25 expressing B cells collected from different lymphoid organs in mice. Samples were stained for various cell surface markers and analysed using flow cytometry. We found that approximately 49% of B cells in bone marrow, 16% in peritoneal cavity, 2% in spleen and 1% in lymph nodes express CD25. In contrast, CD25 expressing B cells were not found in the blood or in Peyer's patches. Phenotypic characterization showed that CD25+ B cells in spleen, lymph nodes and peritoneal cavity have higher expression of AA4.1, CD5, CD69, CD80, CD86, CD122, CD132, IgA, IgG and IgM on their surface in comparison with CD25− B cells. In contrast, expression of IgD and IA‐IE was lower on CD25+ B cells in spleen and lymph nodes. In bone marrow, the expression of CD5, CD80, CD86, CD122, CD132, IgA, IgD and IgM was lower, while the expression of AA4.1, IgG and IA‐IE was increased on CD25+ B cells compared with CD25− B cells. In conclusion, our results indicate that B cells expressing CD25 are phenotypically distinctly different from those that are CD25 negative. Our findings suggest that CD25+ B cells are more prone to efficient antigen presentation and display a more mature phenotype.
Dendritic Cells Therapy Confers a Protective Microenvironment in Murine Pregnancy
Authors: Miranda S. , Litwin S. , Barrientos G. , Szereday L. , Chuluyan E. , Bartho J. S. , Arck P. C. , Blois S. M.
Published: October 2006

The fetal–placental unit is a semi‐allograft and immunological recognition of pregnancy, together with the subsequent response of the maternal immune system, is necessary for a successful pregnancy. Dendritic cells (DC) show a biological plasticity that confers them special characteristics regulating both immunity and tolerance. Therapy employing DC proved to diminish the abortion in the DBA/2J‐mated CBA/J females; however, the underlying mechanisms remain unknown. Here, we evaluated whether DC therapy influences the presence of immunoregulatory populations of cells at the fetal–maternal interface. To address this hypothesis, we analysed the pregnancy‐protective CD8, γδ cell populations as well as transforming growth factor (TGF)‐β1 and progesterone‐induced blocking factor (PIBF) expression at the fetal–maternal interface from abortion‐prone female mice that had previously received adoptive transfer of syngeneic DC. Syngeneic DC therapy induced an increase in the number of CD8 and γδ cells. Additionally, an upregulation of TGF‐β1 and PIBF expression could be detected after DC transfer. We suggest that DC therapy differentially upregulates a regulatory/protective population of cells at the fetal–maternal interface. It is reasonable to assure that this mechanism would be responsible for the lower abortion rate.
The post‐Human Genome Project mindset: race, reliability, and health care
Authors: Kimmelman J
Published: October 2006

The following essay reports on the first session of a 2‐day workshop on genetic diversity and science communication, organized by the Institute of Genetics. I argue that the four talks in this session reflected two different facets of a ‘post‐Human Genome Project (HGP)’ view of human genetics. The first is characterized by an increasing interest in genetic differences. Two speakers – Troy Duster and Jasber Singh – expressed skepticism about one aspect of this trend: an emphasis on race in medicine and genetics. The other two speakers – Kenneth Weiss and Gustavo Turecki – spoke to a second facet of the post‐HGP view: a recognition of the difficulty in translating genetic discovery into medical or public health applications. Though both sets of talks were highly critical of current trends in genetic research, they pulled in opposite directions: one warned about the role of genetics in stabilizing racial categories, while the other lamented the failure of any genetic claims or categories to stabilize at all. I argue that the use of racial categories in medicine seems likely to encounter scientific, medical, and social challenges.
Pediatric lung transplantation: Literature review 2005
Authors: Jerkic P. , Aurora P. , Elliott M. J.
Published: October 2006

Abstract:  This review summarizes important original articles in the field of pediatric lung transplantation that were published in 2005. The review is intended to be comprehensive, but not exhaustive.
IMMORTALITY, HUMAN NATURE, THE VALUE OF LIFE AND THE VALUE OF LIFE EXTENSION
Authors: HORROBIN STEVEN
Published: October 2006

The emerging discourse concerning the desirability of intervention in senescence to achieve radical life extension for persons has featured some striking blurring in traditional liberal and conservative commitments and positions. This affords an opportunity for re‐evaluation of these same. The canonical conservative view of the intrinsic value of life is re‐examined and found primarily to involve a denial of human prerogative, rather than an active underwriting of the value of life extension. A critique is offered of an attempted argument against aging intervention from a proto‐conservative worry about a purported threat to human nature. Immortality is found to be a red herring, but a revealing one. Further, the classic liberal view is examined and found wanting in terms of the gravity of its own commitment to, and fullness of its account of the value of life, and the value of life extension. An analysis of the liberal conception of personhood is proposed that both defines persons necessarily as processes, and demonstrates the inalienable quality of the value of life extension to persons so defined.
BIOETHICS AND DEMOCRACY: COMPETING ROLES OF NATIONAL BIOETHICS ORGANISATIONS
Authors: DODDS SUSAN , THOMSON COLIN
Published: October 2006

In establishing National Bioethics Organisations (NBOs), liberal democracies seek to acknowledge the diversity of strongly held ethical positions and the imperative to engage in public debate about important bioethical decisions. NBOs are typically given a range of responsibilities, including contributing to and stimulating public debate; providing expert opinion on relevant issues for policy deliberations; and developing public policy. The state is now found to have an interest in areas previously thought to be a matter of individual choice. NBOs can provide one way of opening up public debate to allow the diversity of views to be heard in a manner that is well‐informed, articulate and responsive to both expert and ‘lay’ public views. We draw on debates in political theory about democratic decision‐making and on the policy making roles of some key NBOs. We are particularly interested in examining the capacity of NBOs to meet the democratic ideal of effective participation by the public, or citizenry, especially by those who are directly affected by the policies, in the development of effective public policy. We provide a basic framework for policy development involving NBOs that can begin to meet this ideal, a process of ‘contested deliberation’.
THE DATE AND SEQUENCE OF USE OF NEOLITHIC FUNERARY MONUMENTS: NEW AMS DATING EVIDENCE FROM THE COTSWOLD‐SEVERN REGION
Authors: SMITH MARTIN , BRICKLEY MEGAN
Published: October 2006

Summary.  In this paper we discuss 26 new Neolithic AMS dates obtained from human and animal bone from four previously undated funerary monuments in the Cotswold‐Severn region. By strategically targeting particular portions of these skeletal assemblages, a number of valuable inferences are made concerning the extent of variation in apparently co‐existing burial practices both within and between monuments. Of particular interest is the observation that variations in the extent to which interments have become disarticulated cannot necessarily be equated with chronological relationships regarding their deposition. This project has also obtained dates from cremated bone, which establish that the range of funerary treatments in practice during the earlier Neolithic also included cremation. Additionally it is observed that whilst some, apparently primary, deposits may in fact be later insertions, other material in apparently secondary contexts may actually return earlier Neolithic dates.
OFFSHORE FINDS FROM THE BRONZE AGE IN NORTH‐WESTERN EUROPE: THE SHIPWRECK SCENARIO REVISITED
Authors: SAMSON ALICE V.M.
Published: October 2006

Summary.  This article challenges received thinking relating to the interpretation of Bronze Age finds from the seabed in the waters of north‐western Europe, especially the North Sea and Channel area. Metal objects recovered from the sea are traditionally presumed to be the result of shipwrecks. As such, their interpretation as casual, if unfortunate loss is unquestioned. However, abandoning the shipwreck scenario as a remnant of the ‘sacred vs profane’ heuristic, it is suggested that offshore finds could provide insight into deliberate Bronze Age maritime practice, rather than misadventure. Certain patterning in the data of offshore finds, including affinities with hoards on terra firma, urges another interpretive framework – that of considering the sea as a place for deposition. This appeared to be the case particularly in regions which experienced an intensity of maritime interaction, such as the Channel area during the later Bronze Age. From this it is hypothesized that rather than being considered outside the Bronze Age social realm, the sea, especially in the MBA to earlier LBA in the Channel area, was incorporated into Bronze Age cosmology in similar ways to other zones in the landscape.
LATE BRONZE AND IRON AGE LAKE SETTLEMENT IN SCOTLAND AND IRELAND: THE DEVELOPMENT OF THE ‘CRANNOG’ IN THE NORTH AND WEST
Authors: CAVERS GRAEME
Published: October 2006

Summary.  This paper considers the evidence for the origins and development of the lake settlement tradition of Scotland and Ireland in the Late Bronze and Iron Ages. Considering a crannog ‘event horizon’ around the mid‐first millennium BC, dating and structural evidence are compared and contrasted, and the evidence for non‐domestic activity including ritual and votive deposition is contextualized. It is argued that the concurrent appearance of crannogs with the flourish of domestic monumentality in Scotland and Ireland can be seen as a consequence of the fusion of ritual and domestic spheres of life in the later first millennium BC, integrating the themes of architectural monumentality and the Iron Age reverence of water.
Gender‐specific development of nonverbal behaviours and mild depression in adolescence
Authors: Van Beek Yolanda , Van Dolderen Marlies S. M. , Demon Dubas Judith J. S.
Published: October 2006

Background:  Individual differences in depressive symptoms have been linked with social skill deficits in adults and children, yet empirical studies on adolescents are lacking. The present research examines age and gender differences in nonverbal behaviour between mildly depressed and nondepressed (pre‐) adolescents during conversations with an adult (study 1) and a same‐aged peer (study 2). Both studies also examine whether conversation partners respond differently to mildly depressed versus nondepressed (pre)adolescents.

Methods:  Study 1 reports on observations of conversations of 9–15‐year‐old children (n = 122) with a female adult partner. Study 2 reports findings of observations of 12–17‐year‐old adolescents (n = 154) in conversation with same‐age, same‐sex peers.

Results:  Both studies show gender and/or age effects in gazing, smiling and backchannel behaviours that indicate that as adolescents mature they increasingly behave according to gender‐specific display rules. While talking to an adult, depressed (pre‐)adolescents and the adult partner differed in backchannel behaviours. While talking to peers, only depressed adolescent girls showed less gazing towards the partner during listening. Moreover, adolescents smiled less often towards depressed than nondepressed partners.

Conclusions:  Gender‐specific development of nonverbal behaviour may help to understand the development of gender differences in depression in adolescence. Females who fail to exhibit other‐oriented social skills may be particularly at risk for depressive symptoms.
Annotation: What electrical brain activity tells us about brain function that other techniques cannot tell us – a child psychiatric perspective
Authors: Banaschewski Tobias , Brandeis Daniel
Published: October 2006

Background:  Monitoring brain processes in real time requires genuine subsecond resolution to follow the typical timing and frequency of neural events. Non‐invasive recordings of electric (EEG/ERP) and magnetic (MEG) fields provide this time resolution. They directly measure neural activations associated with a wide variety of brain states and processes, even during sleep or in infants. Mapping and source estimation can localise these time‐varying activation patterns inside the brain.

Methods: Recent EEG/ERP research on brain functions in the domains of attention and executive functioning, perception, memory, language, emotion and motor processing in ADHD, autism, childhood‐onset schizophrenia, Tourette syndrome, specific language disorder and developmental dyslexia, anxiety, obsessive‐compulsive disorder, and depression is reviewed.

Results:  Over the past two decades, electrophysiology has substantially contributed to the understanding of brain functions during normal development, and psychiatric conditions of children and adolescents. Its time resolution has been important to measure covert processes, and to distinguish cause and effect.

Conclusions:  In the future, EEG/ERP parameters will increasingly characterise the interplay of neural states and information processing. They are particularly promising tools for multilevel investigations of etiological pathways and potential predictors of clinical treatment response.
Evidence for Two Distinct Forms of Native Glutamic Acid Decarboxylase in Rat Brain Soluble Extract: An Immunoblotting Study
Authors: Legay F. , Henry S. , Tappaz M.
Published: October 2006

Abstract: Immunoblots of the soluble proteins from a rat brain high‐speed supernatant dissociated under reducing conditions showed two monomers (molecular weights, 59,000 and 62,000 ± 2,000) immunolabeled by a glutamic acid decarboxylase (GAD) antiserum. In this extract, a GAD monoclonal antibody trapped the same two monomers, thus confirming that they are both constitutive sub‐units of GAD. Without treatment under reducing conditions, two additional bands were stained by immunoblotting. Their molecular weights were estimated to be 115,000 and 122,000 ± 5,000. These results demonstrate the presence, in rat brain soluble extract, of two distinct forms of native GAD. They further support our previous hypothesis that each form is composed by the homodimeric association of each constitutive subunit through disulfide bridges.
Regional Distribution of Substance P‐ and Thyrotrophin‐Releasing Hormone‐Like Immunoreactivity and Indoleamines in the Rabbit Spinal Cord
Authors: Fone K. C. F. , Bennett G. W. , Marsden C. A.
Published: October 2006

Abstract: The distribution of thyrotrophin‐releasing hormone (TRH), substance P, and the indoleamines [5‐hy‐droxytryptamine (5‐HT) and 5‐hydroxyindoleacetic acid (5‐HIAA)] has been examined in selected regions of the thoracic and lumbar spinal cord of the rabbit using sensitive radioimmunoassays for the first two and HPLC with electrochemical detection for the indoleamines. The levels of TRH‐ and substance P‐like immunoreactivity (TRH‐I and SP‐I, respectively) were greatest in the ventral and dorsal grey matter, respectively. The level of TRH‐I in most thoracic regions was greater than that in equivalent lumbar regions, but the only segmental difference in SP‐I was in the ventral grey matter, where the lumbar segment contained more immunoreactivity. 5‐HT and 5‐HIAA were more evenly distributed than either peptide and showed no segmental variation in levels in equivalent regions, but the ventral grey matter contained significantly higher levels of 5‐HT and had a greater 5‐HT/5‐HIAA ratio than all other regions. The absolute levels and the overall distribution of SP‐I, TRH‐I, and indoleamines in the thoracolumbar cord of the rabbit was very similar to that previously reported in both rats and humans, and the possible functional role of the peptides and indoleamines in spinal neurones is discussed.
Phosphatidylinositol Phosphodiesterase (Phospholipase C) Activity in the Pineal Gland: Characterization and Photoneural Regulation
Authors: Ho Anthony K. , Klein David C.
Published: October 2006

Abstract: Phosphatidylinositol phosphodiesterase (PL‐C) appears to be a key element in the adrenergic regulation of pineal cyclic AMP levels. In the present study, the rat pineal enzyme was characterized using exogenous [3H]phosphati‐dylinositol (0.5 mM) as substrate. Half the enzyme activity was found in the cytosolic fraction, but the highest specific concentration was associated with the membrane fraction. Two pH optima (5.5 and 7.5) of enzyme activity were observed for the membrane fraction but only one in the cyto‐sol fraction (pH 5.5). Enzyme activity in both fractions was Ca2+ dependent. In the case of the membrane protein in pH 7.5, the enzyme activity was sensitive to changes in Ca2+ in the 10–100 nAf range. Addition of an equimolar concentration of phosphatidylinositol 4‐phosphate nearly completely inhibited the hydrolysis of [3H]phosphatidylinositol; other phospholipids (1.0 mM) were less potent. This may reflect our present finding that [3H]phosphatidylinositol 4‐phosphate is a better substrate than [3H]phosphatidylinositol for the enzyme. Stimulus deprivation (2 weeks of constant light or superior cervical ganglionectomy) reduced the cytosolic activity by 30% and had no effect on the membrane‐associated enzyme.