bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Role for Cdk1/Cyclin B in Regulating B-Raf Activation at Mitosis
Authors: Borysov Sergiy I. , Guadagno Thomas M.
Published: April 2008

MAPK activity is important during mitosis for spindle assembly and maintenance of the spindle checkpoint arrest. We previously identified B-Raf as a critical activator of the MAPK cascade during mitosis in Xenopus egg extracts and showed that B-Raf activation is regulated in an M-phase–dependent manner. The mechanism that mediates B-Raf activation at mitosis has not been elucidated. Interestingly, activation of 95-kDa B-Raf at mitosis does not require phosphorylation of Thr-599 and Ser-602 residues (Thr-633 and Ser-636 in Xenopus B-Raf), previously shown to be essential for B-Raf activation by Ras. Instead, we provide evidence for Cdk1/cyclin B in mediating mitotic activation of B-Raf. In particular, Cdk1/cyclin B complexes associate with B-Raf at mitosis in Xenopus egg extracts and contribute to its phosphorylation. Mutagenesis and in vitro kinase assays demonstrated that Cdk1/cyclin B directly phosphorylates B-Raf at Serine-144, which is part of a conserved Cdk1 preferential consensus site (S144PQK). Importantly, phosphorylation of Ser-144 is absolutely required for mitotic activation of B-Raf and subsequent activation of the MAPK cascade. However, substitution of a phospho-mimicking amino acid at Ser-144 failed to produce a constitutive active B-Raf indicating that, in addition of Ser-144 phosphorylation, other regulatory events may be needed to activate B-Raf at mitosis. Taken together, our data reveal a novel cell cycle mechanism for activating the B-Raf/MEK/MAPK cascade.
The RING Domain of cIAP1 Mediates the Degradation of RING-bearing Inhibitor of Apoptosis Proteins by Distinct Pathways
Authors: Cheung Herman H. , Plenchette Stéphanie , Kern Chris J. , Mahoney Douglas J. , Korneluk Robert G.
Published: April 2008

The Inhibitor of Apoptosis proteins (IAPs) are key repressors of apoptosis. Several IAP proteins contain a RING domain that functions as an E3 ubiquitin ligase involved in the ubiquitin-proteasome pathway. Here we investigated the interplay of ubiquitin-proteasome pathway and RING-mediated IAP turnover. We found that the CARD-RING domain of cIAP1 (cIAP1-CR) is capable of down-regulating protein levels of RING-bearing IAPs such as cIAP1, cIAP2, XIAP, and Livin, while sparing NAIP and Survivin, which do not possess a RING domain. To determine whether polyubiquitination was required, we tested the ability of cIAP1-CR to degrade IAPs under conditions that impair ubiquitination modifications. Remarkably, although the ablation of E1 ubiquitin-activating enzyme prevented cIAP1-CR–mediated down-regulation of cIAP1 and cIAP2, there was no impact on degradation of XIAP and Livin. XIAP mutants that were not ubiquitinated in vivo were readily down-regulated by cIAP1-CR. Moreover, XIAP degradation in response to cisplatin and doxorubicin was largely prevented in cIAP1-silenced cells, despite cIAP2 up-regulation. The knockdown of cIAP1 and cIAP2 partially blunted Fas ligand-mediated down-regulation of XIAP and protected cells from cell death. Together, these results show that the E3 ligase RING domain of cIAP1 targets RING-bearing IAPs for proteasomal degradation by ubiquitin-dependent and -independent pathways.
Annotation-Modules: a tool for finding significant combinations of multisource annotations for gene lists
Authors: Hackenberg Michael , Matthiesen Rune
Published: April 2008

Motivation: The ontological analysis of the gene lists obtained from DNA microarray experiments constitutes an important step in understanding the underlying biology of the analyzed system. Over the last years, many other high-throughput techniques emerged, covering now basically all ‘omics’ fields. However, for some of these techniques the generally used functional ontologies might not be sufficient to describe the biological system represented by the derived gene lists. For a more complete and correct interpretation of these experiments, it is important to extend substantially the number of annotations, adapting the ontological analysis to the new emerging techniques.

Results: We developed Annotation-Modules, which offers an improvement over the current tools in two critical aspects. First, the underlying annotation database implements features from many different fields like gene regulation and expression, sequence properties, evolution and conservation, genomic localization and functional categories—resulting in about 60 different annotation features. Second, it examines not only single annotations but also all the combinations, which is important to gain insight into the interplay of different mechanisms in the analyzed biological system.

Availability: http://web.bioinformatics.cicbiogune.es/AM/AnnotationModules.php

Contact:  mlhackenberg@gmail.com
TOPDOM: database of domains and motifs with conservative location in transmembrane proteins
Authors: Tusnády Gábor E. , Kalmár Lajos , Hegyi Hédi , Tompa Péter , Simon István
Published: April 2008

Summary: The TOPDOM database is a collection of domains and sequence motifs located consistently on the same side of the membrane in α-helical transmembrane proteins. The database was created by scanning well-annotated transmembrane protein sequences in the UniProt database by specific domain or motif detecting algorithms. The identified domains or motifs were added to the database if they were uniformly annotated on the same side of the membrane of the various proteins in the UniProt database. The information about the location of the collected domains and motifs can be incorporated into constrained topology prediction algorithms, like HMMTOP, increasing the prediction accuracy.

Availability: The TOPDOM database and the constrained HMMTOP prediction server are available on the page http://topdom.enzim.hu

Contact:  tusi@enzim.hu; lkalmar@enzim.hu
Molecular evolution of the
γ–Herpesvirinae
Authors: McGeoch Duncan J.
Published: April 2008

Genomic sequences available for members of the γ–
Herpesvirinae
allow analysis of many aspects of the group's evolution. This paper examines four topics: (i) the phylogeny of the group; (ii) the histories of γ–herpesvirus–specific genes; (iii) genomic variation of human herpesvirus 8 (HHV–8); and (iv) the relationship between Epstein–Barr virus types 1 and 2 (EBV–1 and EBV–2). A phylogenetic tree based on eight conserved genes has been constructed for eight γ–herpesviruses and extended to 14 species with smaller gene sets. This gave a generally robust assignment of evolutionary relationships, with the exception of murine herpesvirus 4 (MHV–4), which could not be placed unambiguously on the tree and which has evidently experienced an unusually high rate of genomic change. The γ–herpesviruses possess a variable complement of genes with cellular homologues. In the clearest cases these virus genes were shown to have originated from host genome lineages in the distant past. HHV–8 possesses at its left genomic terminus a highly diverse gene (
K1
) and at its right terminus a gene (
K15
) having two diverged alleles. It was proposed that the high diversity of
K1
results from a positive selection on
K1
and a hitchhiking effect that reduces diversity elsewhere in the genome. EBV–1 and EBV–2 differ in their alleles of the
EBNA–2
,
EBNA–3A
,
EBNA–3B
and
EBNA–3C
genes. It was suggested that EBV–1 and EBV–2 may recombine in mixed infections so that their sequences outside these genes remain homogeneous. Models for genesis of the types, by recombination between diverged parents or by local divergence from a single lineage, both present difficulties.
Molecular virology of Epstein–Barr virus
Authors: Bornkamm Georg W. , Hammerschmidt Wolfgang
Published: April 2008

Epstein–Barr virus (EBV) interacts with its host in three distinct ways in a highly regulated fashion: (i) EBV infects human B lymphocytes and induces proliferation of the infected cells, (ii) it enters into a latent phasein vivothat follows the proliferative phase, and (iii) it can be reactivated giving rise to the production of infectious progeny for reinfection of cells of the same type or transmission of the virus to another individual. In healthy people, these processes take place simultaneously in different anatomical and functional compartments and are linked to each other in a highly dynamic steady–state equilibrium. The development of a genetic system has paved the way for the dissection of those processes at a molecular level that can be studiedin vitro, i.e. B–cell immortalization and the lytic cycle leading to production of infectious progeny. Polymerase chain reaction analyses coupled to fluorescent–activated cell sorting has on the other hand allowed a descriptive analysis of the virus–host interaction in peripheral blood cells as well as in tonsillar B cellsin vivo. This paper is aimed at compiling our present knowledge on the process of B–cell immortalizationin vitroas well asin vivolatency, and attempts to integrate this knowledge into the framework of the viral life cyclein vivo.
Dissecting the host response to a γ–herpesvirus
Authors: Doherty Peter C. , Christensen Jan P. , Belz Gabrielle T. , Stevenson Philip G. , Sangster Mark Y.
Published: April 2008

The murine γ–herpesvirus 68 (MHV–68) provides a unique experimental model for dissecting immunity to large DNA viruses that persist in B lymphocytes. The analysis is greatly facilitated by the availability of genetically disrupted (–/–) mice that lack key host–response elements, and by the fact that MHV–68 is a lytic virus that can readily be manipulated for mutational analysis. The mutant virus strategy is being used, for example, to characterize the part playedin vivoby an MHV–68–encoded chemokine–binding protein that may ultimately find an application in human therapeutics. Experiments with various –/– mice and monoclonal antibody depletion protocols have shown very clearly that type I interferons (IFNs) are essential for the early control of MHV–68 replication, while CD4+T cells producing IFN–γ function to limit the consequences of viral persistence. Virus–specific CD8+effectors acting in the absence of the CD4+subset seem initially to control the lytic phase in the lung following respiratory challenge, but are then unable to prevent the reactivation of replicative infection in epithelia and the eventual death of CD4+T–cell–deficient mice. This could reflect the fact that the interaction between the CD8+T cells and the virus–infected targets is partially compromised by the MHV–68 K3 protein, which inhibits antigen presentation by MHC class I glycoproteins. Immunization strategies focusing on the CD8+T–cell response to epitopes expressed during the lytic phase of MHV–68 infection can limit virus replication, but are unable to prevent the establishment of latency. Other experiments with mutant viruses also suggest that there is a disconnection between lytic MHV–68 infection and latency. The massive nonspecific immunoglobulin response and the dramatic expansion of Vβ4+CD8+T cells, which is apparently MHC independent, could represent some sort of ‘smoke screen’ used by MHV–68 to subvert immunity. Although MHV–68 is neither Epstein–Barr virus nor human herpesvirus–8, the results generated from this system suggest possibilities that may usefully be addressed with these human pathogens. Perhaps the main lesson learned to date is that all the components of immunity are likely to be important for the control of these complex viruses.
Preface
Authors: Zeki Semir
Published: April 2008

The maturity of a subject and that of its authors go together, by definition, and yet it is not alwasys that we can celebrate the two together. This special issue of
Philosophical Transactions
provides a rare opportunity. In it we celebrate the coming of age of an important discovery, namely that casual events in the genesis of human cancers can have a viral component. We also celebrate the work of Sir Anthony Epstein, Whose 80the birthday falls on 18 May 2001, and whose brilliant insights led to this dicovery.
Psychophysiological correlates of aggression and violence: an integrative review
Authors: Patrick Christopher J
Published: April 2008

This paper reviews existing psychophysiological studies of aggression and violent behaviour including research employing autonomic, electrocortical and neuroimaging measures. Robust physiological correlates of persistent aggressive behaviour evident in this literature include low baseline heart rate, enhanced autonomic reactivity to stressful or aversive stimuli, enhanced EEG slow wave activity, reduced P300 brain potential response and indications from structural and functional neuroimaging studies of dysfunction in frontocortical and limbic brain regions that mediate emotional processing and regulation. The findings are interpreted within a conceptual framework that draws on two integrative models in the literature. The first is a recently developed hierarchical model of impulse control (externalizing) problems, in which various disinhibitory syndromes including aggressive and addictive behaviours of different kinds are seen as arising from common as well as distinctive aetiologic factors. This model represents an approach to organizing these various interrelated phenotypes and investigating their common and distinctive aetiologic substrates. The other is a neurobiological model that posits impairments in affective regulatory circuits in the brain as a key mechanism for impulsive aggressive behaviour. This model provides a perspective for integrating findings from studies employing different measures that have implicated varying brain structures and physiological systems in violent and aggressive behaviour.
Aggression in young children with concurrent callous–unemotional traits: can the neurosciences inform progress and innovation in treatment approaches?
Authors: Dadds Mark R , Rhodes Tracy
Published: April 2008

Parenting is the ‘clean water’ of healthy psychological development and parenting interventions remain the number one treatment at the individual and community levels for early-onset aggression and antisocial behaviour in children. However, recent progress in child psychopathology research is specifying a number of biological mechanisms that interact with environmental risk to influence pathways into aggression and antisocial behaviour. After a brief review of the parent training literature, we focus on child factors, especially callous–unemotional traits, that parse ‘aggressive’ children into more homogeneous groupings, and then review selected ideas about the origins of aggression coming from the neurosciences (such as neurobehavioural responsivity to emotional stimuli; hypothalamic–pituitary axis abnormalities influencing low cortisol and low serotonin production). We review human and, where relevant, animal models of neurobiological system changes with particular attention to developmental timing and interactions with environmental factors, especially parenting. Based on this innovative research, we then discuss a number of ideas that hold potential for interventions. We conclude that the future will see the development of interventions that aim for synergy between specific biological processes and psychological experiences as they unfold developmentally. The use of
d
-cycloserine in fear extinction and oxytocin in affiliative bonds is used as an example of these futuristic approaches.
Neurobiology and the development of violence: common assumptions and controversies
Authors: Loeber Rolf , Pardini Dustin
Published: April 2008

This paper addresses four common assumptions and related controversies regarding neurobiological factors explaining violence: (i) scholars often assume stability of individual differences in neurobiological factors pertaining to violence, yet much change occurs in aggression/violence during the life course, (ii) individual differences in aggression/violence reflect one or more underlying mechanisms that are believed to have neurobiological origins, yet there is little agreement about which underlying mechanisms apply best, (iii) the development of aggression/violence to some degree can be explained by social, individual, economic and environmental factors, yet it is unclear to what extent neurobiological factors can explain the escalation to, and desistance from, violence over and above social, individual, economic and environmental factors, and (iv) violence waxes and wanes in society over time, yet the explanation of secular differences in violence by means of neurobiological and other factors is not clear. Longitudinal analyses from the Pittsburgh Youth Study are used to illustrate several of these issues.
Evaluation of the GenoType Mycobacteria Direct assay for the simultaneous detection of the Mycobacterium tuberculosis complex and four atypical mycobacterial species in smear-positive respiratory specimens
Authors: Seagar A.-Louise , Prendergast Carmel , Emmanuel F. Xavier , Rayner Alan , Thomson Susan , Laurenson Ian F.
Published: April 2008

A novel, commercially available reverse hybridization assay [GenoType Mycobacteria Direct (GTMD), version 2.0; Hain Lifescience] was evaluated for the direct detection of five clinically relevant mycobacterial species [Mycobacterium tuberculosis complex (MTBC), Mycobacterium avium, Mycobacterium malmoense, Mycobacterium kansasii and Mycobacterium intracellulare] from 54 smear-positive respiratory specimens and the findings were compared with culture results. Three approaches were used for specimen preparation using either whole or ‘split’ sample volumes and N-acetyl-l-cysteine/3 % NaOH or 4 % NaOH as decontamination chemicals. Forty-three out of 52 samples in which RNA amplification was successful gave GTMD results that concurred with the identification of the cultured isolate. All cases of MTBC were detected. Twenty-two samples contained M. tuberculosis complex, seven had M. kansasii, four had M. malmoense, seven contained atypical mycobacteria other than those detectable using the GTMD assay and three specimens contained no viable mycobacteria. The assay is easy to use and can be completed in one working day. Results interpretation is facilitated by the inclusion of an internal amplification control with each sample to allow identification of specimens containing amplification inhibitors. A positive GTMD result will quickly identify patients with MTBC infection or provide specific identification of four other atypical mycobacteria from the same specimen. This allows more rapid drug susceptibility testing, treatment, and public health and infection control decisions.
Post-operative mediastinitis, pleuritis and pericarditis due to Mycoplasma hominis and Ureaplasma urealyticum with a fatal outcome
Authors: García-de-la-Fuente Celia , Miñambres Eduardo , Ugalde Estibaliz , Sáez Ana , Martinez-Martinez Luis , Fariñas M. Carmen
Published: April 2008

Post-sternotomy mediastinitis, although infrequent, is a potentially life-threatening complication of cardiac surgery. We report an unusual case of Mycoplasma hominis and Ureaplasma urealyticum post-surgical mediastinitis with persistent pleural and pericardial effusion. Clinical manifestations and response to therapy are described, and the difficulties of establishing the diagnosis are discussed.
A case of pharyngitis caused by Streptococcus pneumoniae
Authors: Savini Vincenzo , Favaro Marco , Fontana Carla , Consilvio Nicola Pietro , Manna Assunta , Talia Marzia , Catavitello Chiara , Balbinot Andrea , Febbo Fabio , Di Bonaventura Giovanni , Di Giuseppe Nicola , D'Antonio Domenico
Published: April 2008

Throat cultures from an adult pharyngitis patient yielded Streptococcus pneumoniae as a single organism, with a very high bacterial count. The isolate was found to be macrolide and fluoroquinolone resistant, and the same strain was cultured from the patient's denture washing solution. Ceftriaxone therapy, a gradual reduction in the bacterial count and progressive clinical improvement proceeded at the same pace, so we labelled this clinical case as a pneumococcal pharyngitis.
The prevalence of periodontopathogenic bacteria in saliva is linked to periodontal health status and oral malodour
Authors: Kurata Hiroshi , Awano Shuji , Yoshida Akihiro , Ansai Toshihiro , Takehara Tadamichi
Published: April 2008

This study investigated whether an improvement in periodontal health resulted in changes in the prevalence of periodontopathogenic bacteria in saliva and tongue coatings and a reduction in volatile sulfur compounds (VSCs: H2S and CH3SH) linked to oral malodour. The subjects were 35 patients who visited the breath odour clinic of Kyushu Dental College, Japan. Their mean age was 51.2±18.3 years (mean±sd). A clinical examination performed at baseline and 2 months after periodontal treatment assessed VSCs in mouth air using gas chromatography, periodontal probing depth and bleeding on probing (BOP) in all subjects; saliva and tongue coatings were also collected. Genomic DNA was isolated from the samples, and the proportions of five periodontopathogenic bacteria (Porphyromonas gingivalis, Tannerella forsythensis, Treponema denticola, Prevotella intermedia and Prevotella nigrescens) were investigated using quantitative real-time PCR. The subjects were classified into four groups based on the presence of a periodontal pocket of more than 4 mm (PD) and VSCs above the organoleptic threshold level (VSCT) as follows: –PD/–VSCT group, subjects without PD or VSCT; –PD/+VSCT group, those without PD but with VSCT; +PD/–VSCT group, those with PD but without VSCT; and +PD/+VSCT group, those with PD and VSCT. Although the mean PD values in the +PD/–VSCT and +PD/+VSCT groups, BOP in the +PD/+VSCT group, and H2S and CH3SH concentrations in the –PD/+VSCT and +PD/+VSCT groups were greater than in the other groups at baseline, we found no significant difference among the four groups after periodontal treatment. The proportion of periodontopathogenic bacteria in saliva was higher in the +PD/–VSCT and +PD/+VSCT groups than in the –PD/–VSCT and –PD/+VSCT groups at baseline and after treatment, but the proportions of bacteria in saliva after treatment were reduced compared to the baseline. Furthermore, the differences in the proportions of the five target bacteria in the tongue coating were not as apparent as those in saliva at baseline or after treatment. The prevalence of periodontopathogenic bacteria in saliva may reflect periodontal health status and influence VSC levels in mouth air.
A rare presentation of ventriculitis and brain abscess caused by Fusobacterium nucleatum
Authors: Kai Anneke , Cooke Fiona , Antoun Nagui , Siddharthan Chandran , Sule Olajumoke
Published: April 2008

Anaerobic ventriculitis is rare, and usually seen in patients with predisposing factors such as otitis media, mastoiditis, sinusitis or recent neurosurgery. We report what we believe to be the first case of ventriculitis and brain abscess due to Fusobacterium nucleatum infection in a man with no significant predisposing factors. He was successfully treated with antibiotic therapy.
Frequency and expression of mutacin biosynthesis genes in isolates of Streptococcus mutans with different mutacin-producing phenotypes
Authors: Kamiya Regianne Umeko , Höfling José Francisco , Gonçalves Reginaldo Bruno
Published: April 2008

The aim of this study was to analyse the frequency and expression of biosynthesis genes in 47 Streptococcus mutans isolates with different mutacin-producing phenotypes. Detection of the frequency and expression of genes encoding mutacin types I, II, III and IV were carried out by PCR and semi-quantitative RT-PCR, respectively, using primers specific for each type of biosynthesis gene. In addition, a further eight genes encoding putative bacteriocins, designated bsm 283, bsm 299, bsm 423, bsm 1889c, bsm 1892c, bsm 1896, bsm 1906c and bsm 1914, were also screened. There was a high phenotypic diversity; some Streptococcus mutans isolates presented broad antimicrobial spectra against other Streptococcus mutans clinical isolates, including bacteria resistant to common antibiotics, as well as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Streptococcus pyogenes. The expression frequency of the bsm gene was higher than that of the previously characterized mutacins (I–IV). There was no positive correlation between the number of indicator strains inhibited (antimicrobial spectra) and the number of biosynthesis genes expressed (Spearman correlation test, r=−0.03, P>0.05). In conclusion, the high diversity of mutacin-producing phenotypes, associated with high frequency of expression of the biosynthesis genes screened, reveals a broad repertoire of genetic determinants encoding antimicrobial peptides that can act in different combinations.
Identification of hyperinvasive Campylobacter jejuni strains isolated from poultry and human clinical sources
Authors: Fearnley Catherine , Manning Georgina , Bagnall Mary , Javed Muhammad Afzal , Wassenaar Trudy M. , Newell Diane G.
Published: April 2008

Campylobacter jejuni causes gastroenteritis with a variety of symptoms in humans. In the absence of a suitable animal model, in vitro models have been used to study virulence traits such as invasion and toxin production. In this study, 113 C. jejuni isolates from poultry and poultry-related (n=74) environments as well as isolates from human cases (n=39) of campylobacteriosis and bacteraemia were tested for invasiveness using INT 407 cells. The method was sufficiently reproducible to observe a spectrum of invasiveness amongst strains. As a result, strains were classified as low, high and hyper-invasive. The majority of strains (poultry and human) were low invaders (82 % and 88 %, respectively). High invasion was found for 5 % of human strains and 11 % of poultry-related isolates. However, only 1 % of poultry strains were classified as hyperinvasive compared to 13 % of human isolates (P=0.0182). Of those isolates derived from the blood of bacteraemic patients, 20 % were hyperinvasive, though this correlation was not statistically significant. An attempt was made to correlate invasiveness with the presence of seven genes previously reported to be associated with virulence. Most of these genes did not correlate with invasiveness, but gene cj0486 was weakly over-represented, and a negative correlation was observed for the gene ciaB. This trend was stronger when the two genes were analysed together, thus ciaB− cj0486
+ was over-represented in high and hyperinvasive strains, with low invaders more commonly found to lack these genes (P=0.0064).
A Genome-Wide Functional Investigation into the Roles of Receptor-Like Proteins in Arabidopsis
Authors: Wang Guodong , Ellendorff Ursula , Kemp Ben , Mansfield John W. , Forsyth Alec , Mitchell Kathy , Bastas Kubilay , Liu Chun-Ming , Woods-Tör Alison , Zipfel Cyril , de Wit Pierre J.G.M. , Jones Jonathan D.G. , Tör Mahmut , Thomma Bart P.H.J.
Published: April 2008

Receptor-like proteins (RLPs) are cell surface receptors that typically consist of an extracellular leucine-rich repeat domain, a transmembrane domain, and a short cytoplasmatic tail. In several plant species, RLPs have been found to play a role in disease resistance, such as the tomato (Solanum lycopersicum) Cf and Ve proteins and the apple (Malus domestica) HcrVf2 protein that mediate resistance against the fungal pathogens Cladosporium fulvum, Verticillium spp., and Venturia inaequalis, respectively. In addition, RLPs play a role in plant development; Arabidopsis (Arabidopsis thaliana) TOO MANY MOUTHS (TMM) regulates stomatal distribution, while Arabidopsis CLAVATA2 (CLV2) and its functional maize (Zea mays) ortholog FASCINATED EAR2 regulate meristem maintenance. In total, 57 RLP genes have been identified in the Arabidopsis genome and a genome-wide collection of T-DNA insertion lines was assembled. This collection was functionally analyzed with respect to plant growth and development and sensitivity to various stress responses, including susceptibility toward pathogens. A number of novel developmental phenotypes were revealed for our CLV2 and TMM insertion mutants. In addition, one AtRLP gene was found to mediate abscisic acid sensitivity and another AtRLP gene was found to influence nonhost resistance toward Pseudomonas syringae pv phaseolicola. This genome-wide collection of Arabidopsis RLP gene T-DNA insertion mutants provides a tool for future investigations into the biological roles of RLPs.
Structure and dynamics of de novo proteins from a designed superfamily of 4‐helix bundles
Authors: Go Abigail , Kim Seho , Baum Jean , Hecht Michael H.
Published: April 2008

Libraries of de novo proteins provide an opportunity to explore the structural and functional potential of biological molecules that have not been biased by billions of years of evolutionary selection. Given the enormity of sequence space, a rational approach to library design is likely to yield a higher fraction of folded and functional proteins than a stochastic sampling of random sequences. We previously investigated the potential of library design by binary patterning of hydrophobic and hydrophilic amino acids. The structure of the most stable protein from a binary patterned library of de novo 4‐helix bundles was solved previously and shown to be consistent with the design. One structure, however, cannot fully assess the potential of the design strategy, nor can it account for differences in the stabilities of individual proteins. To more fully probe the quality of the library, we now report the NMR structure of a second protein, S‐836. Protein S‐836 proved to be a 4‐helix bundle, consistent with design. The similarity between the two solved structures reinforces previous evidence that binary patterning can encode stable, 4‐helix bundles. Despite their global similarities, the two proteins have cores that are packed at different degrees of tightness. The relationship between packing and dynamics was probed using the Modelfree approach, which showed that regions containing a high frequency of chemical exchange coincide with less well‐packed side chains. These studies show (1) that binary patterning can drive folding into a particular topology without the explicit design of residue‐by‐residue packing, and (2) that within a superfamily of binary patterned proteins, the structures and dynamics of individual proteins are modulated by the identity and packing of residues in the hydrophobic core.
Cysteine 155 plays an important role in the assembly of Mycobacterium tuberculosis FtsZ
Authors: Jaiswal Richa , Panda Dulal
Published: April 2008

The assembly of FtsZ plays an important role in bacterial cell division. Mycobacterium tuberculosis FtsZ (MtbFtsZ) has a single cysteine residue at position 155. We have investigated the role of the lone cysteine residue in the assembly of MtbFtsZ using different complimentary approaches, namely chemical modification by a thiol‐specific reagent 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB) or a cysteine‐chelating agent HgCl2, and site‐directed mutagenesis of the cysteine residue. HgCl2 strongly reduced the polymerized mass of MtbFtsZ while it had no detectable effect on the polymerization of Escherichia coli FtsZ, which lacks a cysteine residue. HgCl2 inhibited the protofilamentous assembly of MtbFtsZ and induced the aggregation of the protein. Further, HgCl2 perturbed the secondary structure of MtbFtsZ and increased the binding of a hydrophobic probe 1‐anilinonaphthalene‐8‐sulfonic acid (ANS) with MtbFtsZ, indicating that the binding of HgCl2 altered the conformation of MtbFtsZ. Chemical modification of MtbFtsZ by DTNB also decreased the polymerized mass of MtbFtsZ. Further, the mutagenesis of Cys‐155 to alanine caused a strong reduction in the assembly of MtbFtsZ. Under assembly conditions, the mutated protein formed aggregates instead of protofilaments. Far‐UV CD spectroscopy and ANS binding suggested that the mutated MtbFtsZ has different conformation than that of the native MtbFtsZ. The effect of the mutation or chemical modification of Cys‐155 on the MtbFtsZ assembly has been explained considering its location in the MtbFtsZ crystal structure. The results together suggest that the cysteine residue (Cys‐155) of MtbFtsZ plays an important role in the assembly of MtbFtsZ into protofilaments.
Conformational gating of dimannose binding to the antiviral protein cyanovirin revealed from the crystal structure at 1.35 Å resolution
Authors: Fromme Raimund , Katiliene Zivile , Fromme Petra , Ghirlanda Giovanna
Published: April 2008

Cyanovirin (CV‐N) is a small lectin with potent HIV neutralization activity, which could be exploited for a mucosal defense against HIV infection. The wild‐type (wt) protein binds with high affinity to mannose‐rich oligosaccharides on the surface of gp120 through two quasi‐symmetric sites, located in domains A and B. We recently reported on a mutant of CV‐N that contained a single functional mannose‐binding site, domain B, showing that multivalent binding to oligomannosides is necessary for antiviral activity. The structure of the complex with dimannose determined at 1.8 Å resolution revealed a different conformation of the binding site than previously observed in the NMR structure of wt CV‐N. Here, we present the 1.35 Å resolution structure of the complex, which traps three different binding conformations of the site and provides experimental support for a locking and gating mechanism in the nanoscale time regime observed by molecular dynamics simulations.
Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E. coli: Enzymatic characterization with crystal structure analysis
Authors: Wu Dalei , Hu Tiancen , Zhang Liang , Chen Jing , Du Jiamu , Ding Jianping , Jiang Hualiang , Shen Xu
Published: April 2008

Alanine racemase (Alr) is an important enzyme that catalyzes the interconversion of L‐alanine and D‐alanine, an essential building block in the peptidoglycan biosynthesis. For the small size of the Alr active site, its conserved substrate entryway has been proposed as a potential choice for drug design. In this work, we fully analyzed the crystal structures of the native, the D‐cycloserine‐bound, and four mutants (P219A, E221A, E221K, and E221P) of biosynthetic Alr from Escherichia coli (EcAlr) and studied the potential roles in substrate orientation for the key residues involved in the substrate entryway in conjunction with the enzymatic assays. Structurally, it was discovered that EcAlr is similar to the Pseudomonas aeruginosa catabolic Alr in both overall and active site geometries. Mutation of the conserved negatively charged residue aspartate 164 or glutamate 165 at the substrate entryway could obviously reduce the binding affinity of enzyme against the substrate and decrease the turnover numbers in both D‐ to L‐Ala and L‐ to D‐Ala directions, especially when mutated to lysine with the opposite charge. However, mutation of Pro219 or Glu221 had only negligible or a small influence on the enzymatic activity. Together with the enzymatic and structural investigation results, we thus proposed that the negatively charged residues Asp164 and Glu165 around the substrate entryway play an important role in substrate orientation with cooperation of the positively charged Arg280 and Arg300 on the opposite monomer. Our findings are expected to provide some useful structural information for inhibitor design targeting the substrate entryway of Alr.
Influence of methylmalonyl‐CoA mutase alleles on resistance to bovine tuberculosis in the European wild boar (Sus scrofa)
Authors: Naranjo V. , Acevedo‐Whitehouse K. , Vicente J. , Gortazar C. , de la Fuente J.
Published: April 2008

An association study was carried out to examine the influence of methylmalonyl‐CoA mutase (MUT) polymorphisms on the susceptibility of a well‐studied wild boar population from southern Spain to develop bovine tuberculosis (bTB). To this end, we examined polymorphisms at a closely linked dinucleotide microsatellite flanking exon 2 of the MUT gene in 37 wild boars with bTB and 36 non‐infected individuals. The microsatellite showed low polymorphism in the studied population, with only three alleles (MUTm‐A, MUTm‐B and MUTm‐C) found, in contrast to the 11 alleles previously reported for domestic pigs. Our case–control study showed that the MUTm‐B allele was associated with disease in a dominant pattern (odds ratio = 3.36; 95% CI = 1.05–10.72; P = 0.04), while the MUTm AA genotype appeared to have a protective effect against bTB infection (odds ratio = 4.33; 95% CI = 1.20–14.96; P = 0.02). Interestingly, infected wild boars heterozygous for MUTm AB are at an advantage (11‐fold) to contain the systemic spread of the disease when compared to other genotypes, implying that a balanced polymorphism may be present in the population. These results strengthen previous observations regarding the importance of the MUT gene on bTB resistance in wild boars and indicate that polymorphisms at this locus will influence the risk of acquiring and maintaining bTB in the studied population.
Do mesenchymal stem cells play a role in vocal fold fat graft survival?
Authors: Lo Cicero V. , Montelatici E. , Cantarella G. , Mazzola R. , Sambataro G. , Rebulla P. , Lazzari Lorenza
Published: April 2008

Abstract.  Objectives: Adipose tissue in vocal fold lipoinjection is currently used to treat patients affected by laryngeal hemiplegia or anatomical defects. The aim of this study has been to evaluate the efficacy of this clinical strategy, by long‐term follow‐up of the patients and to investigate whether the fat samples used to treat them contain a stem cell population with a wide differentiation potential. Materials and methods: Fat samples harvested from 12 patients affected by severe breathy dysphonia who had undergone vocal fold lipoinjection were analysed by immunocytochemistry, by flow cytometry and reverse transcription‐polymerase chain reaction, and the isolated adipose derived mesenchymal stem cells (ADMSCs) were evaluated in order to define their ability to produce soluble factors possibly involved in tissue regeneration, and to differentiate towards different lineages. Results: ADMSCs were efficiently and successfully isolated from all of the samples. They were positive for SSEA‐4, an embryonic marker recently identified on bone marrow MSCs and which could explain their high differentiation plasticity. Molecular analysis showed that these cells also expressed Oct‐4, Runx‐1 and ABCG‐2, which characterize the stem cell state, and a number of other specific lineage markers. Flow cytometry revealed mesenchymal markers expressed on ADMSCs and identified a subpopulation characterized by CD146+/34−/45− cells consistent with perivascular/pericyte‐like cells. Osteogenic, adipogenic and endothelial tissue differentiation were obtained. Conclusions: Our results confirmed the therapeutic efficacy of this clinical approach and showed that adipose tissue, administered to patients in order to restore glottic competence, contains mesenchymal stem cells.
Metformin increases fasting plasma peptide tyrosine tyrosine (PYY) in women with polycystic ovarian syndrome (PCOS)
Authors: Tsilchorozidou Tasoula , Batterham Rachel L. , Conway Gerard S.
Published: April 2008

Objective  The beneficial effects of metformin in patients with type 2 diabetes mellitus (T2DM) and polycystic ovarian syndrome (PCOS) are thought to be in part due to weight reduction. However, the mechanisms by which metformin causes weight loss are unclear. We sought to determine whether circulating levels of the anorectic gut hormone peptide tyrosine tyrosine (PYY) show any correlation with metformin‐induced weight loss.

Design and patients  We examined the acute effects of orally administrated metformin on fasting PYY levels in eight healthy normal‐weight female subjects. Subsequently, we evaluated the effects of 6 months metformin treatment on fasting PYY levels and anthropometric measurements in 20 women with PCOS.

Results  In normal‐weight females 10 days’ metformin treatment increased fasting PYY levels (P < 0·01). Similarly, in PCOS subjects metformin treatment increased fasting PYY concentrations (P < 0·05). In both groups a marked variation in PYY increase in response to metformin was observed. Long‐term metformin treatment was associated with improvements in weight (P < 0·05), BMI (P < 0·05), fasting glucose (P < 0·05) and menstrual frequency (P < 0·01). Interestingly, change in PYY levels were correlated with change in waist circumference (r = 0·55, P < 0·05).

Conclusions  Acute and chronic oral metformin administration increase fasting PYY levels and may contribute to metformin's weight loss effect. Further studies are now required to clarify whether changes in circulating PYY levels in response to metformin treatment can be used to predict which patients will subsequently lose weight long‐term and gain cycle restoration.
Expression and localization of ATP binding cassette (ABC) family of drug transporters in gastric hepatoid adenocarcinomas
Authors: Kamata S , Kishimoto T , Kobayashi S , Miyazaki M
Published: April 2008

Aims:  Hepatoid adenocarcinomas are clinically chemoresistant; however, data concerning the mechanisms of chemoresistance are lacking. ATP‐binding cassette (ABC) transporters play a role in the ATP‐dependent outward efflux of drugs, and their function renders tumour cells multidrug resistant. We assumed that the expression of ABC transporters may be accompanied by hepatic differentiation because most ABC transporters are dominantly expressed in hepatocytes. The aim of this study was to investigate the expression of ABC transporters in hepatoid adenocarcinomas.

Methods and results:  The expression of P‐glycoprotein, multidrug resistant‐associated protein (MRP) 1, MRP2, MRP3 and MRP6 was investigated using immunohistochemistry in 13 cases of gastric hepatoid adenocarcinoma and 32 cases of control adenocarcinoma. P‐glycoprotein was expressed in all cases examined. The positive rates of MRP1 (84.6% versus 21.9%), MRP2 (100% versus 43.8%) and MRP6 (61.5% versus 15.6%) were significantly higher in hepatoid than in control adenocarcinoma. MRP3 was negative in all hepatoid adenocarcinomas. Immunohistochemistry by polyclonal anti‐carcinoembryonic antigen antibody, which detects bile canaliculi, suggested that MRP2 and MRP6 are located on bile canalicular‐like structures in sheet or trabecular nests.

Conclusions:  These results support the hypothesis that ABC transporters participate in the mechanisms of multidrug resistance in hepatoid adenocarcinomas.
Health care costs of persons with newly diagnosed hepatitis C virus: a population‐based, observational study
Authors: Nguyen Thu‐Ha , Jacobs Philip , Hanrahan Anita , Fraser‐Lee Nonie , Wong Winnie , Lee Bonita , Ohinmaa Arto
Published: April 2008

Summary.  The objective of this paper was to conduct an analysis of the health services costs for persons who have been diagnosed with hepatitis C, from the time of diagnosis. Data were based on 1230 persons diagnosed with hepatitis C in 1998 in the Capital Health region of Alberta. Identifiers and dates of diagnosis were sent to Alberta Health and Wellness where records were linked to those of physician visits and billings, as well as hospital (inpatient and outpatient) visit records. Costs were assigned to all visits, and data were analysed for one pre‐ and two post‐diagnosis years. Total cost per person increased from $2630 (Canadian) to $3514 between the pre‐ and first post‐diagnosis year. They then returned to $2694 in the second post‐diagnosis year. Liver‐related costs were a low portion of the total in all periods, though they increased following diagnosis. Mental‐health related costs were the largest component. Observational data present a more balanced picture of the costs of persons with hepatitis C, though most current estimates are not based on such data. Our results indicate that, when analysed within the picture of the entire person, liver‐related costs (which have been the focus of most studies to date) are the tip of the iceberg.
Cell architecture comes to phage biology
Authors: Hendrix Roger
Published: April 2008

Nothing is more iconic of the early days of molecular biology than the image of a bacteriophage infecting an Escherichia coli cell. It is perhaps surprising therefore that more than 50 years later, it is still possible to learn something entirely new and unexpected about how phage infection works, as we see in the paper by Edgar et al. of this issue of Molecular Microbiology. The results give fundamental new insight into the way these viruses infect their hosts and promise to open new windows on the virus–host interactions that have shaped the evolution of both.
Measuring and adjusting the weathering and hydraulic sorting effects for rigorous provenance analysis of sedimentary rocks: a case study from the Jurassic Ashikita Group, south‐west Japan
Authors: OHTA TOHRU
Published: April 2008

The present study examines the provenance of the Jurassic Ashikita Group distributed in south‐west Japan, which is composed of the Idenohana, Kyodomari and Sakamoto Formations. Two geochemical diagrams for provenance analysis were utilized, which incorporate full consideration of compositional modifications resulting from weathering (MFW diagram) and hydraulic sorting processes (SiO2/Al2O3–Na2O/K2O diagram). The MFW diagram delineates weathering trends of sedimentary rocks and allows estimation of the original source rock composition by tracing the weathering trends backwards to an unweathered domain. Weathering trends of the Idenohana and Kyodomari Formations extend backward to the domain of intermediate and felsic igneous rocks. In contrast, sediments of the Sakamoto Formation do not fit into a linear weathering trend, indicating that the source rock cannot be approximated to igneous rocks. On the SiO2/Al2O3–Na2O/K2O diagram, sediments are organized into compositional trends, in which the range reflects compositional variations induced by the hydraulic sorting effect. On this diagram, sediments derived from the igneous and recycled sedimentary provenances can be distinguished by reading the inclination of the trend. By utilizing this principle, source rocks of the Idenohana and Kyodomari Formations are interpreted as igneous rocks and those of the Sakamoto Formation are interpreted as recycled sedimentary rocks. Therefore, these diagrams concurrently estimate the source rock composition through quantifying and adjusting the weathering and sorting effects, and reveal a systematic transition in the provenance of the Ashikita Group. The Idenohana and Kyodomari Formations were supplied chiefly from an igneous provenance, which shifted from intermediate to felsic compositions in stratigraphic order. Whereas, sediments of the Sakamoto Formation were sourced primarily from a recycled sedimentary provenance.
Clinical features and mutational survey of NPHS2 (podocin) in Japanese children with focal segmental glomerulosclerosis who underwent renal transplantation
Authors: Furue Takeki , Hattori Motoshi , Tsukaguchi Hiroyasu , Kitamura Akiko , Oomori Tae , Ogino Daisuke , Nakakura Hyogo , Ashida Akira , Miura Kenichiro , Hisano Masataka , Takahashi Kazuhiro , Chikamoto Hiroko , Akioka Yuko , Sakano Takashi
Published: April 2008

Abstract:  Recurrent FSGS is a major challenge in the field of nephrology. To clarify the role of NPHS2 defects in the pathogenesis of FSGS recurrence, we sequenced all eight exons of NPHS2 in 11 Japanese pediatric FSGS patients with or without post‐transplant recurrence. All patients had biopsy‐proven primary FSGS, had no family history of renal diseases or consanguinity, were steroid‐resistant, and received living‐related renal transplantation. The mean age at onset was 5.0 ± 3.1 yr and mean age at renal transplantation was 10.4 ± 4.1 yr. Mutational analysis of NPHS2 was performed using polymerase chain reaction and direct sequencing. We found a synonymous T/C polymorphism at alanine 318 (GCC to GCT) in seven of 11 patients but no other causative NPHS2 mutations. FSGS recurred immediately after transplant in seven patients, while the remaining four patients had no recurrence for 3.2–5.8 yr. There were no differences between recurrent and non‐recurrent patients in the onset age and the interval from onset to ESRD. In conclusion, we detected no causative NPHS2 mutations in Japanese pediatric FSGS patients with or without post‐transplant recurrence. Further studies on the involvement of other genes are required to better understand recurrent FSGS.