bluuebunny/crossref_metadata_2025 huggingface dataset:
Harp (harmonin‐interacting, ankyrin repeat‐containing protein), a novel protein that interacts with harmonin in epithelial tissues
Authors: Johnston Anne M. , Naselli Gaetano , Niwa Hideo , Brodnicki Thomas , Harrison Leonard C. , Góñez L. Jorge
Published: October 2004

Mutations in the triple PDZ domain‐containing protein harmonin have been identified as the cause of Usher deafness syndrome type 1C. Independently, we identified harmonin in a screen for genes expressed in pancreatic β cells. Using a yeast two‐hybrid assay, we show that the first PDZ domain of harmonin interacts with a novel protein, designated harp for harmonin‐interacting, ankyrin repeat‐containing protein. This interaction was confirmed in an over‐expression system and in mammalian cells, and shown to be mediated by the three C‐terminal amino acids of harp. Harp is expressed in many of the same epithelia as harmonin and co‐localization of native harp and harmonin was demonstrated by confocal microscopy in pancreatic duct epithelium and in a pancreatic β‐cell line. Harp, predicted molecular mass 48 kDa, has a domain structure which includes three ankyrin repeats and a sterile alpha motif. Human harp maps to chromosome 16, and its mouse homologue to chromosome 7. Sequences with similarity to harp include the sans gene, mutations of which are responsible for deafness in the Jackson shaker 2 (js) mutant mouse and in human Usher syndrome type 1G. The functional domain structures of harp and harmonin, their interaction under native conditions and their co‐localization suggest they constitute a scaffolding complex to facilitate signal transduction in epithelia.
A cryptic lysis gene near the start of the Qβ replicase gene in the +1 frame
Authors: Nishihara Tohru , Morisawa Hirokazu , Ohta Norihito , Atkins John F. , Nishimura Yukinobu
Published: October 2004

The maturation/lysis (A2) protein encoded by the group B single‐stranded RNA bacteriophage Qβ mediates lysis of host Escherichia coli cells. We found a frameshift mutation in the replicase (β‐subunit) gene of Qβ cDNA causes cell lysis. The mutant has a single base deletion 73 nucleotides (nt) 3′ from the start of the replicase gene with consequent translation termination at a stop codon 129–131 nt further 3′. The 43‐amino acid C‐terminal part of the 67‐amino acid product encoded by what in WT (wild‐type) is the +1 frame, is rich in basic amino acids This 67‐aa protein can mediate cell lysis whose characteristics indicate that the protein may cause lysis by a different mechanism and via a different target, than that caused by the A2 maturation/lysis protein. Synthesis of a counterpart of the newly discovered lysis product in wild‐type phage infection would require a hypothetical ribosomal frameshifting event. The lysis gene of group A RNA phages is also short, 75 codons in MS2, and partially overlaps the first part of their equivalently located replicase gene, raising significant evolutionary implications for the present finding.
Structural basis for distinct roles of Lys63‐ and Lys48‐linked polyubiquitin chains
Authors: Tenno Takeshi , Fujiwara Kenichiro , Tochio Hidehito , Iwai Kazuhiro , Morita E. Hayato , Hayashi Hidenori , Murata Shigeo , Hiroaki Hidekazu , Sato Mamoru , Tanaka Keiji , Shirakawa Masahiro
Published: October 2004

Ubiquitination, a modification in which single or multiple ubiquitin molecules are attached to a protein, serves as a signalling function that controls a wide variety of cellular processes. To date, two major forms of polyubiquitin chain have been functionally characterized, in which the isopeptide bond linkages involve Lys48 or Lys63. Lys48‐linked polyubiquitin tagging is mostly used to target proteins for degradation by the proteasome, whereas Lys63‐linked polyubiquitination has been linked to numerous cellular events that do not rely on degradative signalling via the proteasome. Apparently linkage‐specific conformations of polyubiquitin chains are important for these cellular functions, but the structural bases distinguishing Lys48‐ and Lys63‐linked chains remain elusive. Here, we report NMR and small‐angle X‐ray scattering (SAXS) studies on the intersubunit interfaces and conformations of Lys63‐ and Lys48‐linked di‐ and tetraubiquitin chains. Our results indicate that, in marked contrast to Lys48‐linked chains, Lys63‐linked chains are elongated molecules with no stable non‐covalent intersubunit interfaces and thus adopt a radically different conformation from that of Lys48‐linked chains.
Testosterone replacement therapy – perceptions of recipients and partners
Authors: Dunning Trisha L. , Ward Glenn M.
Published: October 2004

Background.  The androgenic hormones are important determinants of sexual behaviour in men. Testosterone replacement is important treatment for pituitary disease to maintain normal functioning. Although the physical effects of testosterone replacement have been well documented, little is known about the effects on relationships, particularly from the point of view of the sexual partners of men receiving testosterone replacement.

Aims.  This paper reports a study exploring the perceptions of testosterone replacement on well‐being and sexual functioning.

Methods.  Semi‐structured interviews were conducted with five men receiving testosterone implants (recipients), their permanent partners, and five recipients without partners. Recipient serum testosterone concentration was measured at 0, 1 and 4 months after testosterone implantation.

Results.  The three groups reported similar effects of testosterone on well‐being and sexual functioning. Recipient and partner ratings were also similar. Strength was less affected by decreasing testosterone concentration than energy in men with partners, but both strength and energy declined in men without partners. Decreased testosterone levels had a statistically significantly different effect on libido at time zero between men with and without partners (P < 0·015) and on ability to sustain an erection, but the ability to achieve an erection persisted over the 6 months in both male groups. Intercourse frequency increased from once per week at time 0 to ≥3 per week between 1 and 4 months after implant in men with partners. There were important effects of testosterone deficiency on general and sexual relationships, and these differed between men with partners and those without.

Conclusions.  Testosterone has important physical and psychological benefits that may be related to the age at which testosterone replacement commences and the indications for its use. The small sample size may limit the ability to generalize the findings outside the study.
Breast health information needs of women from minority ethnic groups
Authors: Watts Tessa , Merrell Joy , Murphy Fiona , Williams Angela
Published: October 2004

Background.  For women from minority ethnic groups to make informed decisions about their health, and particularly about whether to participate in breast cancer screening programmes, access to a range of appropriately designed high quality, culturally‐specific and sensitive health information is needed.

Aims.  Through a critical review of the literature, this paper aims to determine the breast health and breast cancer screening information needs of women from minority ethnic groups and to discuss the implications of cultural difference for nurses in relation to the development and dissemination of health information.

Methods.  A critical review of the research literature published in English between 1996 and 2002 was conducted. Electronic and the relevant Cochrane Collaboration databases were searched using a range of search terms to retrieve literature specifically relevant to the aims of the review. The use of personal contacts and posting a request for information on the mailing list at minority-ethnic-health@jiscmail.ac.uk facilitated the retrieval of grey literature. All references retrieved were entered on a bibliographic database. The title and abstract of each was examined to assess it for inclusion in the review.

Findings.  There was little published information about specific breast cancer screening information needs from the perspective of women from minority ethnic groups. In comparison with the indigenous population, the information needs of people from minority ethnic groups differ in relation to their cultural beliefs and values and the effects of these on health care practices. Inadequate knowledge about breast health and breast cancer screening may be a consequence of the provision of insufficient or culturally inappropriate information.

Conclusions.  There is a dearth of research highlighting breast health and breast cancer screening information needs of women from minority ethnic groups. In providing information, their needs appear to have been an ‘add on’. Health care professionals’ lack of understanding about cultural beliefs, values and knowledge, together with racial stereotyping and misconceptions about cancer in minority ethnic groups, pose challenges to information dissemination. Health care professionals need to work collaboratively with women from minority ethnic groups, identifying culturally‐specific beliefs and values about breast cancer, breast cancer risk and screening, in order to develop appropriate and acceptable information and dissemination strategies.
Postoperative quality of life among patients with thyroid cancer
Authors: Huang Sheng‐Miauh , Lee Chen‐Hsen , Chien Li‐Yin , Liu Hsueh‐Erh , Tai Chen‐Jeng
Published: October 2004

Background.  Thyroid cancer patients have a survival rate of over 90% after thyroidectomy. However, quality of life with a generic focus has rarely been studied in thyroid cancer survivors.

Aim.  This paper reports a study to examine and describe factors associated with quality of life among patients with thyroid cancer after operation.

Methods.  Adult patients who had a thyroidectomy for cancer between January 1999 and June 2001 in a Taiwan city participated in the study. Telephone interviews was completed with 146 (67%) of those 218 eligible, asking about sociodemographic variables, disease/treatment characteristics and social support. Quality of life was measured by the Chinese version of the Quality of Life Index. Multivariate analyses were performed using multiple linear regressions.

Result.  The regression model showed that patients at 19–36 months after operation had lower quality of life compared with those within 18 months of operation. Current symptoms of fatigue and chills were negatively associated with quality of life. Those who rated the impact of operational scar on activities as ‘high’ had lower quality of life scores. Social support from families and friends had positive effects on quality of life. The model r2 was 48·1%.

Conclusions.  Nurses could improve quality of life among patients with thyroid cancer by strengthening their social support and educating them in self‐management of uncomfortable symptoms. Surgeons should consider types of operation in which the scar would be less likely to influence the patients’ activities.
Practice nurses’ intentions to use clinical guidelines
Authors: Puffer Suezann , Rashidian Arash
Published: October 2004

Background.  Evidence indicates that, although nurses are increasingly using clinical guidelines to ensure higher quality of care, there is a wide variance in their adherence to them. The utility of the Theory of Planned Behaviour (TPB) has not been previously investigated in explaining this variance in community nursing.

Aim.  This paper reports a study whose primary aim was to examine the utility of the TPB in explaining variations in practice nurses’ intentions to offer smoking cessation advice in accordance with coronary heart disease guidelines.

Methods.  A cross‐sectional survey using a postal questionnaire was carried out. A 52‐item questionnaire was administered to 48 practice nurses in England. The questionnaire was designed to assess the components of the TPB, and included measures of intentions to offer smoking cessation advice, self‐reported past behaviour, attitudes, subjective norms, perceived behavioural controls (PBCs), behavioural beliefs and evaluations, normative beliefs and motivation to comply, and control beliefs and evaluations.

Results.  The TPB explained up to 40% of variance in intentions to offer smoking cessation advice. Attitudes and PBCs were the most important predictors of intention. Among other elements of the TPB, indirect attitudes and indirect PBCs made significant positive contributions to explaining variance in intention.

Discussion.  Future trials of interventions to increase practice nurses’ adherence to clinical guidelines could attempt to address the elements identified in this study as important factors. Further studies are required to examine the utility of the TPB in predicting practice nurses’ behaviour.
The ethics of conducting a co‐operative inquiry with vulnerable people
Authors: Tee Stephen R. , Lathlean Judith A.
Published: October 2004

Background. Mental health services users have been calling for greater participation in clinical research. Participation in this context means research ‘with’ rather than ‘on’ groups of people. Conducting a co‐operative inquiry involving the participation of vulnerable individuals as co‐researchers, in particular those with a history of mental health problems, places an obligation on researchers to articulate and justify sound ethical procedures.

Aims. The aim of this paper is to consider how the ethical issues encountered when conducting participative research with vulnerable people can be addressed in the implementation of a co‐operative inquiry with users of mental health services.

Method. The study was based on personal reflection and a critical review of associated literature obtained from a database search using Boolean logic.

Findings. The findings, presented under the headings of the four prima facie moral principles, suggest the need for researchers using participative approaches to demonstrate the humanistic attributes required for engaging and working with people over a period of time. These include building and maintaining trusting relationships, assessing competence to participate, managing interpersonal and group dynamics and making complex collaborative decisions about participants’ continued participation in a study.

Conclusions. When using a co‐operative inquiry approach involving vulnerable individuals, researchers need to demonstrate clearly how a balance between autonomy and paternalism will be achieved, how risks will be anticipated and managed and how fairness will be maintained throughout all procedures. Researchers using participative approaches need to have developed a level of personal insight and self‐awareness through access to supervision which focuses on sources of unintended manipulation and interpersonal dynamics that may arise at the inception of a study and throughout its course. Researchers and ethics committees have a shared responsibility to ensure that vulnerable people are appropriately engaged to maintain the advancement of user knowledge which informs nursing practice.
From SARS to strategic actions reframing systems
Authors: Macdonald Marilyn T.
Published: October 2004

Background.  The developed world responds to new and re‐emerging diseases through the discovery of medications. Disease can be transmitted around the world in a day, but the development of medications does not occur at this rate. The world has one environment and the focus in health care must be on identifying factors in this environment that coalesce to produce disease.

Aim.  The aim of this paper is to introduce the integrative model of environmental health and explore its potential to illuminate the Toronto SARS experience.

Discussion.  SARS affected people on three continents in a matter of days. Response to this new disease varied from one area to another and was dependent upon the level of integration of health services and communication across services. The present focus of the health care system is on treating the results of disease rather than the causative factors. Reacting to a new disease had grave social and economic consequences. The time for a new global environmental approach to health is now. The Toronto SARS experience was examined using the integrative model of environmental health and the upstream perspective as exemplars to interrupt the traditional approach to disease. All health care providers share the responsibility to learn about and to understand how our environment creates disease. This knowledge comes through research on topics such as; chemicals, pesticides, soil erosion, killing of forests, contamination of water, destabilization of climate, and social disruption from wars.

Conclusions.  Health care systems in the developed world continue to focus on the treatment of disease. A global ecological initiative for an integrated disease prevention system must be negotiated among nations.
Quality monitoring of nurse telephone triage: pilot study
Authors: Richards David A. , Meakins Joan , Tawfik Jane , Godfrey Lesley , Dutton Evelyn , Heywood Phil
Published: October 2004

Background.  Nurse telephone triage is a popular way of managing workload in primary care and has been shown to be as safe as general practitioner (GP)‐delivered alternatives. No studies have tested a routine method of assessing the quality of decision‐making by triage nurses in primary care.

Aims.  The aim of this paper is to describe a study to evaluate accuracy and utility of audiotaping telephone consultations to assess the quality of decisions made by practice nurses triaging same day appointment requests in routine practice.

Method.  An observational study was carried out using external assessment by GPs and nurse practitioners of triage quality in 218 audiotaped nurse telephone triage consultations.

Results.  There were high levels of agreement between triage nurses and assessors on identifying the presenting problem. However, there were only moderate levels of agreement between GPs and nurse assessors both on level of information sought (kappa = 0·434) and appropriateness of outcome (kappa = 0·614). Assessors rated information‐gathering as poor in 19·3% of calls (95% confidence interval 14·6 to 25·0), and seven (3·2%) consultations were rated as potentially dangerous (95% confidence interval 1·6 to 6·5). A 1% sample of all consultations would have a probability of 0·48 of identifying unsafe consultations and 0·99 probability of identifying consultations with poor information‐gathering.

Conclusions.  External assessment of triage quality may be of only moderate accuracy and reliability. Nonetheless, considerable information can be gleaned from assessing audiotapes to assess consultation quality. Even where nurses are generally highly skilled and competent, a 1% review of triage consultations would be sufficient to identify their information‐gathering learning needs in relation to patients’ presenting problems. Audio‐taped consultations could be an important part of clinical governance strategies.
Distinctive genetic features exhibited by the Y‐family DNA polymerases in Bacillus subtilis
Authors: Duigou Stéphane , Ehrlich S. Dusko , Noirot Philippe , Noirot‐Gros Marie‐Françoise
Published: October 2004

Translesional DNA polymerases form a large family of structurally related proteins, known as the Y‐polymerases. Bacillus subtilis encodes two Y‐polymerases, referred herewith as Pol Y1 and Pol Y2. Pol Y1 was expressed constitutively and did not mediate UV mutagenesis. Pol Y1 overexpression increased spontaneous mutagenesis. This effect depended on Pol Y1 polymerase activity, Pol Y1 interaction with the β‐clamp, and did not require the presence of the RecA protein. In addition, Pol Y1 overexpression delayed cell growth at low temperature. The growth delay was mediated by Pol Y1 interaction with the β‐clamp but not by its polymerase activity, suggesting that an excess of Pol Y1 in the cell could sequester the β‐clamp. In contrast, Pol Y2 was expressed during the SOS response, and, in its absence, UV‐induced mutagenesis was abolished. Upon Pol Y2 overproduction, both UV‐induced and spontaneous mutagenesis were stimulated, and both depended on the Pol Y2 polymerase activity. However, UV mutagenesis did not appear to require the interaction of Pol Y2 with the β‐clamp whereas spontaneous mutagenesis did. In addition, Pol Y2‐mediated spontaneous mutagenesis required the presence of RecA. Together, these results show that the regulation and the genetic requirements of the two B. subtilis Y‐polymerases are different, indicating that they fulfil distinct biological roles. Remarkably, Pol Y1 appears to exhibit a mutator activity similar to that of Escherichia coli Pol IV, as well as an E. coli UmuD‐related function in growth delay. Pol Y2 exhibits an E. coli Pol V‐like mutator activity, but probably acts as a single polypeptide to bypass UV lesions. Thus, B. subtilis Pol Y1 and Pol Y2 exhibit distinctive features from the E. coli Y‐polymerases, indicating that different bacteria have adapted different solutions to deal with the lesions in their genetic materiel.
The midcell replication factory in Bacillus subtilis is highly mobile: implications for coordinating chromosome replication with other cell cycle events
Authors: Migocki Margaret D. , Lewis Peter J. , Wake R. Gerry , Harry Elizabeth J.
Published: October 2004

During vegetative growth, rod‐shaped bacterial cells such as Escherichia coli and Bacillus subtilis divide precisely at midcell. It is the Z ring that defines the position of the division site. We previously demonstrated that the early stages of chromosome replication are linked to midcell Z ring assembly in B. subtilis and proposed a direct role for the centrally located replication factory in masking and subsequently unmasking the midcell site for Z ring assembly. We now show that the replication factory is significantly more scattered about the cell centre than the Z ring in both vegetative cells and outgrown spores of B. subtilis. This finding is inconsistent with the midcell replication factory acting as a direct physical block to Z ring assembly. Time‐lapse experiments demonstrated that the lower precision of replication factory positioning results from its high mobility around the cell centre. Various aspects of this mobility are presented and the results are discussed in the light of current views on the determinants of positional information required for accurate chromosome segregation and cell division.
Characterization of a lipoprotein, NilC, required by Xenorhabdus nematophila for mutualism with its nematode host
Authors: Cowles Charles E. , Goodrich‐Blair Heidi
Published: October 2004

Xenorhabdus nematophila is a γ‐proteobacterial mutualist of an insect‐pathogenic nematode, Steinernema carpocapsae. X. nematophila requires nilC, a gene predicted to encode an outer membrane lipoprotein of unknown function, for colonization of its nematode host. Characterization of NilC, described here, demonstrated it is a 28 kDa lipoprotein directed to the periplasm by an N‐terminal signal sequence. Lipidation and processing of NilC occurs by a mechanism that is conserved in proteobacteria. This work also showed NilC is membrane associated and oriented towards the periplasm of X. nematophila and is produced as an outer membrane‐associated protein when expressed in Escherichia coli. Expression analyses revealed that nilC transcription is directly or indirectly repressed by Lrp, and this regulatory link may explain the nematode mutualism defect of a previously identified lrp::Tn5 mutant. An lrp::Tn5 mutant produces an additional nilC transcript, not observed in wild‐type cells growing in vitro, and produces ≈ 75‐fold more nilC than wild‐type cells in late stationary phase. These fundamental characterizations of nilC expression and nilC localization and processing events have provided firm bases for understanding the role of this colonization factor in the X. nematophila/S. carpocapsae microbe–host interaction.
Ss‐LrpB, a novel Lrp‐like regulator of Sulfolobus solfataricus P2, binds cooperatively to three conserved targets in its own control region
Authors: Peeters Eveline , Thia‐Toong Thia‐Lin , Gigot Daniel , Maes Dominique , Charlier Daniel
Published: October 2004

Ss‐LrpB, a novel Lrp‐like DNA‐binding protein from the hyperthermophilic crenarchaeon Sulfolobus solfataricus, was shown to bind cooperatively to three regularly spaced targets in its own control region, with as consensus the 15 bp palindrome 5′‐TTGYAW WWWWTRCAA‐3′. Binding to the border sites occurred with high affinity; the target in the middle proved to be a low affinity site which is stably bound only when both flanking sites are occupied. Ss‐LrpB contacts two major groove segments and the intervening minor groove of each site, all aligned on one face of the helix. The operator shows intrinsic bending and is increasingly deformed upon binding of Ss‐LrpB to one, two and three targets. Complex formation relies therefore on DNA conformability, protein–DNA and protein–protein contacts. Mobility‐shift assays and in gel footprinting indicate that Ss‐LrpB and the transcription factors TATA‐box binding protein (TBP) and transcription factor B (TFB) can bind simultaneously to the control region. Based on these findings we present a model for the construction of the higher order nucleoprotein complexes and a hypothesis for the autoregulatory process. The latter is based on the concentration‐dependent formation of distinct complexes exhibiting different stoichiometries and conformations, which could positively and negatively affect promoter activity.
The elusive malaria sporozoite in the mammalian host
Authors: Baldacci Patricia , Ménard Robert
Published: October 2004

Malaria infection is initiated when Plasmodium sporozoites are injected into a host during the bite of an infected mosquito. In the mammal, the sporozoite must rapidly reach an intravacuolar niche within a hepatocyte, where it will generate the parasite stage that invades red blood cells and causes the symptoms of the disease. Herein, we describe our understanding of the way in which sporozoites travel from the site of the mosquito bite to the liver, arrest in the liver, cross the sinusoidal barrier and eventually gain access to hepatocytes. We also highlight some of the recent advances in our understanding of these processes at the molecular level.
Calcium signalling in bacteria
Authors: Dominguez Delfina C.
Published: October 2004

Whereas the importance of calcium as a cell regulator is well established in eukaryotes, the role of calcium in prokaryotes is still elusive. Over the past few years, there has been an increased interest in the role of calcium in bacteria. It has been demonstrated that as in eukaryotic organisms, the intracellular calcium concentration in prokaryotes is tightly regulated ranging from 100 to 300 nM. It has been found that calcium ions are involved in the maintenance of cell structure, motility, transport and cell differentiation processes such as sporulation, heterocyst formation and fruiting body development. In addition, a number of calcium‐binding proteins have been isolated in several prokaryotic organisms. The characterization of these proteins and the identification of other factors suggest the possibility that calcium signal transduction exists in bacteria. This review presents recent developments of calcium in bacteria as it relates to signal transduction.
Analysis of the N‐terminal DNA binding domain of the IS30 transposase
Authors: Nagy Zita , Szabó Mónika , Chandler Michael , Olasz Ferenc
Published: October 2004

IS30 is the founding member of a large family of widely spread bacterial insertion sequences with closely related transposases. The N‐terminal end of the IS30 transposase had been shown to retain sequence‐specific DNA binding activity and to protect the IS30 terminal inverted repeats. Structural predictions revealed the presence of a helix–helix–turn–helix motif (H–HTH2) which, in the case of IS30, is preceded by an additional helix–turn–helix motif (HTH1). Analysis of deletion and point mutants in this region revealed that both motifs are important for IS30 transposition. IS30 exhibits two types of insertion specificity preferring either a 24 bp palindromic hot‐spot (GOHS) or sequences resembling its ends [left and right terminal inverted repeat (IRL and IRR)]. Results are presented suggesting that the HTH1 region is required for GOHS targeting and interferes with the inverted repeat (IR) targeting. On the other hand, H–HTH2 appears to be required for both. The binding activities of the mutant proteins to the terminal IS30 IRs as measured by gel retardation correlated well with these results. Furthermore, close inspection of the H–HTH2 region revealed significant amino acid identity with a similar predicted secondary structure carried by the transcriptional regulator FixJ of Sinorhizobium meliloti and involved in FixJ binding to its target sequence. This suggests that FixJ and IS30 transposase share similar sequence‐specific DNA binding mechanisms.
Communication between Eσ54, promoter DNA and the conserved threonine residue in the GAFTGA motif of the PspF σ54‐dependent activator during transcription activation
Authors: Bordes Patricia , Wigneshweraraj Siva R. , Chaney Matthew , Dago Angel E. , Morett Enrique , Buck Martin
Published: October 2004

Conversion of Eσ54 closed promoter complexes to open promoter complexes requires specialized activators which are members of the AAA (ATPases Associated with various cellular Activities) protein family. The ATP binding and hydrolysis activity of Eσ54 activators is used in an energy coupling reaction to remodel the Eσ54 closed promoter complex and to overcome the σ54‐imposed block on open complex formation. The remodelling target for the AAA activator within the Eσ54 closed complex includes a complex interface contributed to by Region I of σ54, core RNA polymerase and a promoter DNA fork junction structure, comprising the Eσ54 regulatory centre. One σ54 binding surface on Eσ54 activators is a conserved sequence known as the GAFTGA motif. Here, we present a detailed characterization of the interaction between Region I of σ54 and the Escherichia coli AAA σ54 activator Phage shock protein F. Using Eσ54 promoter complexes that mimic different conformations adopted by the DNA during open complex formation, we investigated the contribution of the conserved threonine residue in the GAFTGA motif to transcription activation. Our results suggest that the organization of the Eσ54 regulatory centre, and in particular the conformation adopted by the σ54 Region I and the DNA fork junction structure during open complex formation, is communicated to the AAA activator via the conserved T residue of the GAFTGA motif.
Candida albicans lacking the frataxin homologue: a relevant yeast model for studying the role of frataxin
Authors: Santos Renata , Buisson Nicole , Knight Simon A. B. , Dancis Andrew , Camadro Jean‐Michel , Lesuisse Emmanuel
Published: October 2004

We cloned the CaYFH1 gene that encodes the yeast frataxin homologue in Candida albicans. CaYFH1 was expressed in Δyfh1 Saccharomyces cerevisiae cells, where it compensated for all the phenotypes tested except for the lack of cytochromes. Double ΔCayfh1/ΔCayfh1 mutant had severe defective growth, accumulated iron in their mitochondria, lacked aconitase and succinate dehydrogenase activity and had defective respiration. The reductive, siderophore and haem uptake systems were constitutively induced and the cells excreted flavins, thus behaving like iron‐deprived wild‐type cells. Mutant cells accumulated reactive oxygen species and were hypersensitive to oxidative stress, but not to iron. Cytochromes were less abundant in mutants than in wild‐type cells, but this did not result from defective haem synthesis. The low cytochrome concentration in mutant cells was comparable to that of iron‐deprived wild‐type cells. Mitochondrial iron was still available for haem synthesis in ΔCayfh1/ΔCayfh1 cells, in contrast to S. cerevisaeΔyfh1 cells. CaYFH1 transcription was strongly induced by iron, which is consistent with a role of CaYfh1 in iron storage. Iron also regulated transcription of CaHEM14 (encoding protoporphyrinogen oxidase) but not that of CaHEM15 (encoding ferrochelatase). There are thus profound differences between S. cerevisiae and C. albicans in terms of haem synthesis, cytochrome turnover and the role of frataxin in these processes.
An in vivo inducible gene of Pseudomonas aeruginosa encodes an anti‐ExsA to suppress the type III secretion system
Authors: Ha Un‐Hwan , Kim Jaewha , Badrane Hassan , Jia Jinghua , Baker Henry V. , Wu Donghai , Jin Shouguang
Published: October 2004

We have previously reported on the isolation of in vivo inducible genes of Pseudomonas aeruginosa using IVET system. One of such genes isolated from burn mouse infection model encodes a short open reading frame with unknown function. In this study, we demonstrate that this gene product specifically suppresses the expression of type III secretion genes in P. aeruginosa, thus named PtrA (Pseudomonas type III repressor A). A direct interaction between the PtrA and type III transcriptional activator ExsA was demonstrated, suggesting that its repressor function is probably realized through inhibition of the ExsA protein function. Indeed, an elevated expression of the exsA compensates the repressor effect of the PtrA. Interestingly, expression of the ptrA is highly and specifically induced by copper cation. A copper‐ responsive two‐component regulatory system, copR‐copS, has also been identified and shown to be essential for the copper resistance in P. aeruginosa as well as the activation of ptrA in response to the copper signal. Elevated expression of the ptrA during the infection of mouse burn wound suggests that P. aeruginosa has evolved tight regulatory systems to shut down energy‐expensive type III secretion apparatus in response to specific environmental signals, such as copper stress.
Enterococcus faecalis pheromone‐responsive protein PrgX: genetic separation of positive autoregulatory functions from those involved in negative regulation of conjugative plasmid transfer
Authors: Kozlowicz Briana K. , Bae Taeok , Dunny Gary M.
Published: October 2004

The pCF10 plasmid in Enterococcus faecalis transfers from donor cells to recipients upon induction via peptide pheromone. Two plasmid‐encoded negative regulators produced from the same transcript, PrgX protein and Qa RNA, repress conjugation genes in uninduced donor cells. PrgX positively autoregulates production of both itself and mature Qa RNA, and is believed to repress the prgQ promoter in a pheromone‐sensitive fashion. Previous analysis of PrgX was complicated because mutations in prgX affecting regulation of conjugation also disrupted PrgX autoregulation, suggesting the two functions might be inseparable. In this study, we isolated 14 single amino acid substitutions in PrgX that reduced or eliminated repression of prgQ, without affecting autoregulation or DNA binding. PrgX was shown to bind to its cognate pheromone, cCF10, and most of the mutations lowered the affinity of PrgX for cCF10. Dimerization was affected by five of the mutations and the data indicate that it is required, but insufficient for pheromone induction. We propose a new model for the mechanism used by PrgX for regulation of the prgQ promoter, PrgX autoregulation, and Qa RNA processing.
A regulation cascade controls expression of Porphyromonas gingivalis fimbriae via the FimR response regulator
Authors: Nishikawa Kiyoshi , Yoshimura Fuminobu , Duncan Margaret J.
Published: October 2004

Little is known about how Porphyromonas gingivalis, a Gram‐negative oral anaerobe, senses environmental changes, and how such information is transmitted to the cell. The production of P. gingivalis surface fimbriae is regulated by FimS–FimR, a two component signal transduction system. Expression of fimA, encoding the fimbrilin protein subunit of fimbriae, is positively regulated by the FimR response regulator. In this study we investigated the molecular mechanisms of FimR regulation of fimA expression. Comparative transcription profiling of fimR wild‐type and mutant strains shows that FimR controls the expression of several genes including five clustered around the fimA locus. Chromatin immunoprecipitation assays and electrophoretic mobility shift assays identify and confirm that FimR binds to the promoter region of the first gene in the fimA cluster. Gene expression analyses of mutant strains reveal a transcriptional cascade involving multiple steps, with FimR activating expression of the first gene of the cluster that encodes a key regulatory protein.
Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium loti strain R7A VirB/D4 type IV secretion system
Authors: Hubber Andree , Vergunst Annette C. , Sullivan John T. , Hooykaas Paul J. J. , Ronson Clive W.
Published: October 2004

The symbiosis island of Mesorhizobium loti strain R7A contains genes with strong similarity to the structural vir genes (virB1‐11; virD4) of Agrobacterium tumefaciens that encode the type IV secretion system (T4SS) required for T‐DNA transfer to plants. In contrast, M. loti strain MAFF303099 lacks these genes but contains genes not present in strain R7A that encode a type III secretion system (T3SS). Here we show by hybridization analysis that most M. loti strains contain the VirB/D4 T4SS and not the T3SS. Strikingly, strain R7A vir gene mutants formed large nodules containing bacteroids on Leucaena leucocephala in contrast to the wild‐type strain that formed only uninfected tumour‐like structures. A rhcJ T3SS mutant of strain MAFF303099 also nodulated L. leucocephala, unlike the wild type. On Lotus corniculatus, the vir mutants were delayed in nodulation and were less competitive compared with the wild type. Two strain R7A genes, msi059 and msi061, were identified through their mutant phenotypes as possibly encoding translocated effector proteins. Both Msi059 and Msi061 were translocated through the A. tumefaciens VirB/D4 system into Saccharomyces cerevisiae and Arabidopsis thaliana, as shown using the Cre recombinase Reporter Assay for Translocation (CRAfT). Taken together, these results suggest that the VirB/D4 T4SS of M. loti R7A plays an analogous symbiotic role to that of T3SS found in other rhizobia. The heterologous translocation of rhizobial proteins by the Agrobacterium VirB/D4 T4SS is the first demonstration that rhizobial effector proteins are translocated into plant cells and confirms functional conservation between the M. loti and A. tumefaciens T4SS.
The PAK family kinase Cla4 is required for budding and morphogenesis in Ustilago maydis
Authors: Leveleki Leonora , Mahlert Michael , Sandrock Björn , Bölker Michael
Published: October 2004

The phytopathogenic basidiomycete Ustilago maydis displays a dimorphic switch between budding growth of haploid cells and filamentous growth of the dikaryon. In a screen for mutants affected in morphogenesis and cytokinesis, we identified the serine/threonine protein kinase Cla4, a member of the family of p21‐activated kinases (PAKs). Cells, in which cla4 has been deleted, are viable but they are unable to bud properly. Instead, cla4 mutant cells grow as branched septate hyphae and divide by contraction and fission at septal cross walls. Delocalized deposition of chitinous cell wall material along the cell surface is observed in cla4 mutant cells. Deletion of the Cdc42/Rac1 interaction domain (CRIB) results in a constitutive active Cla4 kinase, whose expression is lethal for the cell. cla4 mutant cells are unable to induce pathogenic development in plants and to display filamentous growth in a mating reaction, although they are still able to secrete pheromone and to undergo cell fusion with wild‐type cells. We propose that Cla4 is involved in the regulation of cell polarity during budding and filamentation.
Differential regulation of ACC synthase genes in cold‐dependent and ‐independent ripening in pear fruit
Authors: EL‐SHARKAWY I. , JONES B. , GENTZBITTEL L. , LELIÈVRE J.‐M. , PECH J. C. , LATCHÉ A.
Published: October 2004

Late pear cultivars such as Passe‐Crassane (PC) require a long chilling treatment before they are capable of ripening. Early cultivars such as Old‐Home (OH) have no cold prerequisite. The regulation of 1‐aminocyclopropane‐1‐carboxylic acid synthase (ACS) genes was studied in OH, PC and in OH × PC hybrids in order to determine the role of this gene family in the cold requirement. Of the seven Pc‐ACS cDNAs isolated, four (Pc‐ACS1a/b and Pc‐ACS2a/b) showed differential expression associated with the cold requirement. Pc‐ACS1a transcripts accumulated throughout the cold treatment and, with Pc‐ACS2a, during ripening of cold‐dependent cultivars. Pc‐ACS1b and Pc‐ACS2b were detected only during ripening of cold‐independent genotypes. Furthermore, Pc‐ACS2a transcript accumulation was negatively regulated by ethylene, whereas Pc‐ACS2b was positively regulated by the hormone. Pc‐ACS3, 4 and 5 transcript accumulation was similar in all genotypes. Genetic analyses of OH, PC, and 22 OH × PC progenies demonstrated that late, cold‐dependent cultivars were homozygous for Pc‐ACS1a and 2a whereas early, cold‐independent cultivars were heterozygous for Pc‐ACS1(a/b) and homozygous for Pc‐ACS2b. A model is presented in which differences in Pc‐ACS alleles and gene expression between cold‐ and non‐cold‐requiring pears are critical in determining the ripening behaviour of the cultivars.
Does oxygen limit nitrogenase activity in soybean exposed to elevated CO2?
Authors: CEN Y.‐P. , LAYZELL D. B.
Published: October 2004

Soybean (Glycine max L. Merr) plants grown under control (360 µmol mol−1) or elevated CO2 concentration (800 µmol mol−1) from 33 to 42 d after sowing were assayed for various components of in vivo nitrogenase activity to test the hypothesis that increasing carbohydrate supply to nodules would increase the potential (i.e. O2 saturated) nitrogenase activity and impose a more severe O2 limitation on both nodule metabolism and total nitrogenase activity. Within 51 h of elevated CO2 treatment, significant increases relative to control plants were seen in total nitrogenase activity expressed per plant. After 6 d of elevated CO2, the total nitrogenase activity per plant was 18% higher than that in control. This was attributed to an initial increase in nodule size, and a subsequent increase in nodule number following plant exposure to elevated CO2. However, after 9 d of elevated CO2, the potential and total nitrogenase activities per gram nodule dry weight were lower, not higher than corresponding values in plants in the control treatment. These results did not support the hypothesis. It was concluded that the metabolic capacity of the control nodules were not limited by carbohydrate supply, at least at the assay temperatures employed here.
Acclimation of a terrestrial plant to submergence facilitates gas exchange under water
Authors: MOMMER L. , PEDERSEN O. , VISSER E. J. W.
Published: October 2004

Flooding imposes stress upon terrestrial plants since it severely hampers gas exchange rates between the shoot and the environment. The resulting oxygen deficiency is considered to be the major problem for submerged plants. Oxygen microelectrode studies have, however, shown that aquatic plants maintain relatively high internal oxygen pressures under water, and even may release oxygen via the roots into the sediment, also in dark. Based on these results, we challenge the dogma that oxygen pressures in submerged terrestrial plants immediately drop to levels at which aerobic respiration is impaired. The present study demonstrates that the internal oxygen pressure in the petioles of Rumex palustris plants under water is indeed well above the critical oxygen pressure for aerobic respiration, provided that the air‐saturated water is not completely stagnant. The beneficial effect of shoot acclimation of this terrestrial plant species to submergence for gas exchange capacity is also shown. Shoot acclimation to submergence involved a reduction of the diffusion resistance to gases, which was not only functional by increasing diffusion of oxygen into the plant, but also by increasing influx of CO2, which enhances underwater photosynthesis.
Differential expression of dehydrin genes in wild barley, Hordeum spontaneum, associated with resistance to water deficit
Authors: SUPRUNOVA T. , KRUGMAN T. , FAHIMA T. , Chen G. , SHAMS I. , KOROL A. , NEVO E.
Published: October 2004

Dehydrin gene (Dhn) expression is associated with plant response to dehydration. The aim of the present study was to investigate the association of differential expression of Dhn genes (Dhn 1, 3, 5, 6, and 9) with drought tolerance found in wild barley (Hordeum spontaneum). Tolerant and sensitive genotypes were identified from Israeli (Tabigha microsite) and Jordanian (Jarash and Waddi Hassa) populations (based on scoring of water loss rate of 390 genotypes). The five Dhn genes were up‐regulated by dehydration in resistant and sensitive wild barley genotypes. Notably, differences between resistant and sensitive genotypes were detected, mainly in the expression of Dhn1 and Dhn6 genes, depending on the duration of dehydration stress. Dhn1 tended to react earlier (after 3 h) and higher (12 h and 24 h) in resistant compared to sensitive genotypes. The level of expression of Dhn6 was significantly higher in the resistant genotypes at the earlier stages after stress. However, after 12 and 24 h Dhn6 expression was relatively higher in sensitive genotypes. The present results may indicate that these genes have some functional role in the dehydration tolerance in wild barley. The authors suggest that the observed differences of Dhn expression in wild barley, originating from different micro‐ and macro ecogeographic locations, may be the result of adaptive edaphic and climatic selective pressures.
Distribution, developmental and stress responses of antioxidant metabolism in Malus
Authors: DAVEY M. W. , FRANCK C. , KEULEMANS J.
Published: October 2004

A comprehensive study was carried out to examine the interactions between the two major hydrophilic antioxidants l‐ascorbate (vitamin C, l‐AA), and glutathione (γ‐glutamyl cysteinylglycine, GSH), and other antioxidant pools in tissues of Malus, to identify factors affecting steady‐state cellular concentrations. We show that in Malus, each tissue type has a characteristic and different l‐AA/GSH ratio and that in fruit, exocarp (epidermal) tissue acclimated to high light has higher l‐AA levels but lower GSH levels than shaded (green) areas. Maturing seeds were characterized by the highest concentrations of GSH and a highly oxidized l‐AA pool. It is demonstrated that fruit seeds are capable of l‐AA biosynthesis, but that this occurs exclusively by means of the Smirnoff–Wheeler pathway. By contrast, foliar tissue was also able to synthesize l‐AA using uronic acid substrates. Unlike the fruit of some other plant species however, the remaining fruit tissues are incapable of de novol‐AA biosynthesis. The observed differences in the steady‐state concentrations of l‐AA and GSH and the capacity to withstand stress in fruit, were also independent of the rates of uptake of photosynthate or of l‐AA, but were correlated with the protective effect provided by phenolic compounds in these tissues. During development and maturation, l‐AA and GSH levels in apple fruit declined steadily while foliar levels remained essentially constant throughout. However there was no apparent relationship between the free sugar contents of the fruit and antioxidant concentrations.