bluuebunny/crossref_metadata_2025 huggingface dataset:
Activated Vegfr2/kdr Pathway In Tumour Cells And Tumour Associated Vessels Of Colorectal Cancer
Authors: Giatromanolaki A. , Koukourakis M. I. , Sivridis E. , Chlouverakis G. , Vourvouhaki E. , Turley H. , Harris A. L. , Gatter K. C. ,   for the ‘Tumour and Angiogenesis Research Group’
Published: September 2007

Background  Vascular endothelial cell growth factor (VEGF) acts by phosphorylating specific tyrosine kinase receptors on endothelial cell membrane promoting angiogenesis. The study of the activation status of VEGF receptors in human malignancies has recently become feasible by means of specific monoclonal antibodies recognising the phosphorylated form of these receptors.

Materials and methods  In the current study, we investigate the expression of the phosphorylated VEGFR2/KDR receptor in normal colon and colorectal adenocarcinomas in parallel with histopathological parameters, prognosis and the expression of the ‘hypoxia inducible factor’ HIF1α.

Results  pVEGFR2/KDR was weakly expressed in the normal colon, but it was expressed strongly in the cytoplasm and nuclei of cancer cells and in the tumour associated vasculature, mainly at the invading tumour edge. pVEGFR2/KDR expression in cancer cells was significantly associated with a tumour diameter > 6 cm (P = 0·04), poor histological differentiation (P = 0·004) and with high ÇÉF1α expression (P = 0·05). High pVEGFR2/KDR expressing vascular density was significantly related with a high VEGF and HIF1α expression in cancer cells (P = 0·02 and 0·03, respectively). This was also related significantly to high pVEGFR2/KDR expression in cancer cells. In multivariate analysis, the most significant predictors for death were lympho‐vascular invasion (P < 0·001) followed by VEGF (P = 0·014), node status (P = 0·015), standard vascular density (P = 0·022) and necrosis (P = 0·032).

Conclusions  pVEGFR2 receptors are largely expressed in colon cancer cells and intratumoural vasculature. As VEGF targeting agents enter the clinical practice, the role of monoclonal antibodies recognising the phosphorylated form of VEGF receptors as predictors of response to targeted therapies should be sought in clinicopathological trials.
Markers of inflammation and disuse in vastus lateralis of chronic obstructive pulmonary disease patients
Authors: Crul T. , Spruit M. A. , Gayan‐Ramirez G. , Quarck R. , Gosselink R. , Troosters T. , Pitta F. , Decramer M.
Published: September 2007

Background  Disuse and/or local inflammation in the muscle cannot be excluded as potential influences for the decreased muscle force in patients hospitalised due to an acute chronic obstructive pulmonary disease (COPD) exacerbation. This study aims to compare expression levels of markers of disuse (insulin‐like growth factor‐1 (IGF‐I), MyoD and myogenin) and inflammation [interleukin‐6 (IL‐6), IL‐8 and tumour necrosis factor‐alpha (TNF‐α)] in the muscle of hospitalised and stable COPD patients and healthy elderly.

Material and methods  Muscle biopsies (m. vastus lateralis) were taken in 14 hospitalised COPD patients (aged 68 ± 8), 11 clinically stable COPD patients (aged 68 ± 9) and seven healthy subjects (aged 70 ± 7) to analyse local mRNA expression levels of IL‐6, IL‐8, TNF‐α, IGF‐I and protein expression levels of IGF‐I, MyoD and myogenin. Relationships of these expression levels with lung and skeletal muscle function were investigated.

Results  IGF‐I mRNA and MyoD protein levels were significantly lower in hospitalised patients compared to healthy subjects. MyoD protein levels were positively related to quadriceps force. Muscle IL‐6 and IL‐8 expression in hospitalised patients was similar compared to stable patients and healthy subjects and was not related to expression levels of muscle markers of disuse or quadriceps force. Muscle TNF‐α and myogenin were not detected.

Conclusion  Decreased expression levels of muscle IGF‐I and MyoD in hospitalised patients suggest a potential influence of disuse in the increased muscle weakness during an acute COPD exacerbation. This study did not find any evidence supporting local inflammation via IL‐6, IL‐8 and/or TNF‐α in the vastus lateralis muscle of COPD patients.
Hypoxanthine decreases equilibrative type of adenosine transport in lymphocytes from Lesch–Nyhan patients
Authors: Prior C. , Torres R. J. , Puig J. G.
Published: September 2007

Background  Lesch‐Nyhan (LN) syndrome is associated with deficient hypoxanthine‐guanine phosphoribosyltransferase (HPRT) activity, but the connection between the aberrant purine metabolism and the neurological symptoms remains largely unknown. The aim of this study was to determine adenosine transporter subtypes affected by HPRT deficiency and by the associated hypoxanthine excess.

Materials and methods  Nucleoside transporter types (depending on their sodium dependence and 10 µm nitrobenzylthioinosine, NBTI, sensitivity) involved in adenosine transport were compared between peripheral blood lymphocytes (PBL) obtained from nine LN patients, PBLLN (2–21 years) and from nine controls, PBLC (2–23 years) under basal conditions and after 25 µm hypoxanthine incubation.

Results  We found four types of adenosine transporters in PBL: equilibrative and concentrative transporters that are either sensitive (ENT1 or cs) or insensitive (ENT2 or ci) to NBTI. Adenosine ENT1 uptake was the predominant transporter in both PBLC (55%) and PBLLN (46%). Under basal conditions no significant differences were found in adenosine transport between PBLC and PBLLN. Incubation of PBL with 25 µm hypoxanthine markedly decreased total adenosine transport in both cell types. Hypoxanthine affected equilibrative transport (mainly ENT2 type) in PBLLN and PBLC. Only in PBLC was concentrative transport affected by hypoxanthine. Expressions of human (h) ENT1 and hENT2 mRNA were not significantly modified by hypoxanthine incubation in PBLC.

Conclusions  This study contributes to further knowledge of the defective adenosine transport found in PBLLN. Increased hypoxanthine levels, similar to those reported in HPRT deficient patients, reduced adenosine uptake by 32% in PBLLN as compared to normal transport.
Role of soil constituents in fixation of soluble Zn, Cu, Ni and Cd added to soils
Authors: Buekers J. , Van Laer L. , Amery F. , Van Buggenhout S. , Maes A. , Smolders E.
Published: September 2007

Slow immobilization of trace metals in soil, termed ‘fixation’, affects their natural attenuation but it is still unclear which reactions occur. Twenty‐eight soils were selected to assess the role of Fe oxides and carbonates on fixation of Cu, Cd, Zn and Ni. Soils included samples from 2 toposequences (Vietnam, Spain) and 13 European topsoils with different soil characteristics (pH 3.4–7.7). Samples were amended with 250 mg Zn kg−1, 100 mg Cu kg−1, 80 mg Ni kg−1 and 2.5 mg Cd kg−1 as metal salts and incubated for 850 days. Fixation was measured as the increase of the fraction of added metals that were not isotopically exchangeable. Fixation increased with time and was, averaged over all the soils, 43% (Cu), 41% (Zn), 41% (Ni) and 28% (Cd) after 850 days. Metal fixation within samples from each toposequence was generally positively related to total Fe oxide concentration (Fed) for Zn, Ni and Cd. However, the fixation of Cd, Zn and Ni was mainly explained by pH and not by Fed when considering all soils. Fixation of Zn and Cd in soils with pH >7.0 increased with increasing concentrations of carbonates at initial ageing times. Fixed fractions of Zn, Ni and Cd were significantly released when experimentally removing 50% of carbonates by acidification. Fixation of Cu was most poorly related to soil properties. Our data suggest that fixation of Cd, Zn and Ni is related to a pH‐dependent diffusion into oxides and that of Cd and Zn also to diffusion and/or coprecipitation in carbonates. Fixation of Ni at neutral pH may also be related to stabilization of precipitates that form readily in soil.
Allergic inflammatory reaction is involved in necrosis of human pulmonary dirofilariasis
Authors: Araya J , Kawabata Y , Tomichi N , Kaneko K , Hayashi K , Iwabuchi K , Terasaki Y , Kawashima T , Watanabe M
Published: September 2007

Aims:  To determine whether, in view of the massive inflammatory cell infiltration and the rounded rather than wedge‐shaped character of pulmonary lesions in dirofilariasis, the inflammatory response against the worm contributes to the coagulative necrosis, in addition to an ischaemic process.

Methods and results:  The histopathological features of 13 resected dirofilariasis cases with well‐defined nodules ranged from 10 to 30 mm were analysed. On routine histology and using immunohistochemistry, the peripheral encapsulating wall showed mild to severe infiltration of eosinophils, lymphocytes and plasma cells and a histiocytic reaction in all cases, often with necrotic eosinophils seen within the necrosis (84.6%) and inflammatory changes in the adjacent lung (38.5%). The CD4+ lymphocyte count (80.8 ± 33.4) was greater than that of CD8+ lymphocytes (24.5 ± 16.9) in the central necrosis and vice versa in the wall. In the necrotic regions, disruption of the pulmonary artery (61.5%) and extravasation of the torn worm (23.1%) could be seen.

Conclusions:  These findings indicate that an allergic inflammatory reaction, mediated by eosinophils and lymphocytes, is involved in the formation of the dirofilarial necrotizing granuloma rather than infarction caused simply by embolism.
Changes of periodontal parameters following apical surgery: a prospective clinical study of three incision techniques
Authors: Von Arx T. , Vinzens‐Majaniemi T. , Bürgin W. , Jensen S. S.
Published: September 2007

Aim  To evaluate periodontal changes following apical surgery, and to relate changes to the type of incision and to the type of restoration present at the gingival margin (GM).

Methodology  Periodontal parameters [probing depth (PD), level of GM and clinical attachment, plaque and bleeding indices] were recorded at baseline and 1 year following apical surgery. The periodontal changes were calculated and assessed with respect to the incision technique (intrasulcular incision, papilla base incision and submarginal incision), as well as to the presence and type of a restoration margin in contact with the gingiva.

Results  One hundred and eighty‐four teeth could be evaluated. No significant differences between the three incision techniques were found regarding changes in PDs and plaque index over time. However, significant differences between the intrasulcular and submarginal incisions were found for changes in levels of GM and clinical attachment. For example, with the intrasulcular incision, there was a mean recession of 0.42 mm at buccal sites, whereas using the submarginal incision there was a gain of 0.05 mm. No statistically significant influence could be demonstrated for the presence and type of restoration margins, or the smoking habit of the patient.

Conclusion  The type of incision was found to affect changes significantly in periodontal parameters within an observation period of 1 year following apical surgery, whereas the restoration margin and smoking habit did not prove to have any significant effect.
The origins and time of appearance of focal testicular dysgenesis in an animal model of testicular dysgenesis syndrome: evidence for delayed testis development?
Authors: Hutchison Gary R. , Sharpe Richard M. , Mahood I. Kim , Jobling Matt , Walker Marion , McKinnell Chris , Mason J. Ian , Scott Hayley M.
Published: September 2007

A testicular dysgenesis‐like syndrome is induced in rats by fetal exposure to di(n‐butyl) phthalate (DBP). A key feature of this is the formation of focal dysgenetic areas comprising malformed seminiferous cords/tubules and intratubular Leydig cells (ITLC), but how and why these arise remains unclear. The present study has used combinations of cell‐specific markers and immunohistochemistry to address this. The results show that focal dysgenetic areas and ITLC first appear postnatally at 4–10 days of age, but this only occurs in treatment groups in which formation of fetal Leydig cell aggregation is induced between e17.5 and e21.5. Extreme variability in the formation and size of the Leydig cell aggregates probably accounts for the equally extreme variation in occurrence and size of focal dysgenetic areas postnatally. DBP‐induced fetal Leydig cell aggregation traps Sertoli and other cells within the aggregates, but it is unclear why this happens nor why cords fail to form prenatally in these cell mixtures but do elsewhere in the fetal testis. The present studies show that differentiation of the fetal Leydig cells is drastically delayed at e15.5 after DBP exposure, which may be indicative of a wider delay in testis cell development and organisation, and this might account for some of the unexplained findings.
A National Health Service Hospital's cardiac rehabilitation programme: a qualitative analysis of provision
Authors: O'Driscoll Jamie M , Shave Robert , Cushion Christopher J
Published: September 2007

Aim.  This paper reports a study examining the effectiveness of a London National Health Service Trust Hospital's cardiac rehabilitation programme, from the perspectives of healthcare professionals and patients.

Background.  Cardiovascular disease is the world's leading cause of death and disability. Substantial research has reported that, following a cardiac event, cardiac rehabilitation can promote recovery, improve exercise capacity and patient health, reduce various coronary artery disease risk factors and subsequently reduce hospitalization costs. Despite these findings and the introduction of the National Service Framework for Coronary Heart Disease, there is wide variation in the practice, management and organization of cardiac rehabilitation services.

Methods.  A purposeful sample of three postmyocardial infarction patients registered on the selected hospital's cardiac rehabilitation programme, coupled with 11 healthcare professionals were selected. The patients acted as individual case studies. The authors followed all three patients through phase III of their cardiac rehabilitation programme. The research attempted to explore the roles and procedures of a London hospital's cardiac rehabilitation programme through an interpretative framework involving qualitative research methods. Participant observation and in‐depth semi‐structured interviews were the instruments used to collect data.

Findings.  Whilst the healthcare professionals were enthusiastic about coronary heart disease prevention, the London NHS trust hospital's cardiac rehabilitation programme had several barriers, which reduced the programme's success and prevented it from achieving National Service Framework targets. The barriers were complex and mainly included service‐related factors, such as lack of professional training, weak communication between primary and secondary care and confused roles and identities.

Conclusion.  Although the study has immediate relevance for the local area, it highlighted issues of more general relevance to cardiac rehabilitation and secondary prevention programme development, such as communication and role and identity perceptions in a multi‐professional working environment and the need to develop a formal training programme for cardiac rehabilitation healthcare professionals.

Relevance to clinical practice.  The results of this study highlight the need for increased investment, improved planning and the introduction of a comprehensive training programme for healthcare practitioners in cardiac rehabilitation. Implementation of these actions may reduce many of the service limitations and barriers that currently surround cardiac rehabilitation programmes.
A nurse‐led cardiac rehabilitation programme improves health behaviours and cardiac physiological risk parameters: evidence from Chengdu, China
Authors: Jiang Xiaolian , Sit Janet W , Wong Thomas KS
Published: September 2007

Aim.  The aim of this study was to examine the effect of a cardiac rehabilitation programme on health behaviours and physiological risk parameters in patients with coronary heart disease in Chengdu, China.

Background.  Epidemiological studies indicate a dose‐, level‐ and duration‐dependent relationship exists between cardiac behavioural and physiological risks and coronary heart disease incidence as well as subsequent cardiac morbidity and mortality. Cardiac risk factor modification has become the very primary goal of modern cardiac rehabilitation programmes.

Design methods.  A randomized controlled trial was conducted. Coronary heart disease patients (n = 167) who met the sampling criteria in two tertiary medical centres in Chengdu, south‐west China, were randomly assigned to either an intervention group (the cardiac rehabilitation programme) or control group (the routine care). The change of health behaviours (walking performance, step II diet adherence, medication adherence, smoking cessation) and physiological risk parameters (serum lipids, blood pressure, body weight) were assessed to evaluate the programme effect.

Results.  Patients in the intervention group demonstrated a significantly better performance in walking, step II diet adherence, medication adherence; a significantly greater reduction in serum lipids including triglyceride, total cholesterol, low‐density lipoprotein; and significantly better control of systolic and diastolic blood pressure at three months. The majority of these positive impacts were maintained at six months. The effect of the programme on smoking cessation, body weight, serum high‐density lipoprotein, was not confirmed.

Conclusions.  A cardiac rehabilitation programme led by a nurse can significantly improve the health behaviours and cardiac physiological risk parameters in coronary heart disease patients. Nurses can fill significant treatment gaps in the risk factor management of patients with coronary heart disease.

Relevance to clinical practice.  This study raises attention regarding the important roles nurses can play in cardiac rehabilitation and the unique way for nurses to meet the rehabilitative care needs of coronary heart disease patients. Furthermore, the hospital–home bridging nature of the programme also created a model for interfacing the acute care and community rehabilitative care.
The Pleistocene easternmost distribution in Eurasia of the species associated with the Eemian Palaeoloxodon antiquus assemblage
Authors: PUSHKINA DIANA
Published: September 2007

The Palaeoloxodon antiquus large‐mammal assemblage was typical of the late middle and late Pleistocene interglacials in Europe. This review examines the assemblage's origins, dispersal and cohesiveness in Eurasia.

During the more climatically equable middle‐Pleistocene periods, the Palaeoloxodon assemblage (or closely related) species occurred across central Eurasia almost simultaneously. In Central and Western Europe, these species responded to climatic changes together as an unvarying interglacial assemblage, whereas in Eastern Europe and Siberia, they occurred in diverging assemblages. The boundary of the Palaeoloxodon assemblage can be drawn roughly from Poland to Romania.

In Central and Western Europe this interglacial assemblage last occurred during the Eemian. During this period many of the Palaeoloxodon assemblage species may also have co‐occurred in south‐eastern Europe and, except for Bubalus murrensis and Hippopotamus amphibius, further eastwards. The extinct species of the Palaeoloxodon assemblage disappeared in Siberia and Central Asia prior to Europe and the Caucasus whereas the extant species were already present in their modern distribution areas.

A quantitative study of faunal associations across Eurasia, following much‐needed comprehensive systematic reviews, would further elucidate the patterns of faunal change associated with local and global climatic changes during the middle to late Pleistocene.
Fire and ice: volcanic and glacial impacts on the phylogeography of the New Zealand forest fern Asplenium hookerianum
Authors: SHEPHERD LARA D. , PERRIE LEON R. , BROWNSEY PATRICK J.
Published: September 2007

In the Southern Hemisphere there has been little phylogeographical investigation of forest refugia sites during the last glacial. Hooker's spleenwort, Asplenium hookerianum, is a fern that is found throughout New Zealand. It is strongly associated with forest and is a proxy for the survival of woody vegetation during the last glacial maximum. DNA sequence data from the chloroplast trnL‐trnF locus were obtained from 242 samples, including c. 10 individuals from each of 21 focal populations. Most populations contained multiple, and in many cases unique, haplotypes, including those neighbouring formerly glaciated areas, while the predominant inference from nested clade analysis was restricted gene flow with isolation by distance. These results suggest that A. hookerianum survived the last glacial maximum in widespread populations of sufficient size to retain the observed phylogeography, and therefore that the sheltering woody vegetation must have been similarly abundant. This is consistent with palynological interpretations for the survival in New Zealand of thermophilous forest species at considerably smaller distances from the ice sheets than recorded for the Northern Hemisphere. Eastern and central North Island populations of A. hookerianum were characterized by a different subset of haplotypes to populations from the remainder of the country. A similar east–west phylogeographical pattern has been detected in a diverse array of taxa, and has previously been attributed to recurrent vulcanism in the central North Island.
Origins and population genetics of weedy red rice in the USA
Authors: LONDO J. P. , SCHAAL B. A.
Published: September 2007

Weedy red rice (Oryza sativa spontonea) is a persistent and problematic weed of rice culture worldwide. A major hypothesis for the mechanism of production of this weed in South and Southeast Asia is hybridization between cultivated rice (Oryza sativa) and wild rice (Oryza rufipogon). However, weedy red rice can often be found outside the range of O. rufipogon leaving questions on the origin and process behind weedy rice infestations. In the USA, weedy red rice was first documented as early as 1846 and has continued to affect rice production areas. In this study, we attempt to identify the origin and population structure of weedy red rice sampled from the USA using both DNA sequence data from a neutral nuclear locus as well as microsatellite genotype data. Results suggest that two major accessions of weedy rice exist, strawhull and blackhull, and these forms may both hybridize with the cultivated rice of the USA, O. sativa japonica. Using population assignment of multilocus genotype signatures with principal component analysis and structure, an Asian origin is supported for US weedy rice. Additionally, hybridization between strawhull and blackhull varieties was inferred and may present the opportunity for the production of new weedy forms in the future.
Ecological annotation of genes and genomes through ecological genomics
Authors: LANDRY CHRISTIAN R. , AUBIN‐HORTH NADIA
Published: September 2007

Ecological genomics is a research field that aims to determine how a genome or a population of genomes interacts with its environment across ecological and evolutionary timescales. This matter was the central theme of the symposium on Ecological Genomics that took place at the First meeting of the Canadian Society for Ecology and Evolution, held at the University of Toronto in May 2007. Through their research on a diverse array of organisms, the various speakers illustrated how ecology and evolution benefit from genomics, and indirectly how genomics can benefit from evolutionary ecology.
Rates of evolution of avirulence phenotypes and DNA markers in a northwest European population of Puccinia striiformis f. sp. tritici
Authors: HOVMØLLER MOGENS S. , JUSTESEN ANNEMARIE F.
Published: September 2007

The effects of evolutionary processes in fungal pathogen populations may occur more rapidly and display larger effects in agricultural systems than in wild ecosystems because of human involvement by plant breeding and crop management. In this study, we analysed the rate of evolution in three lineages of a northwest European population of a biotrophic and asexual reproduced fungal pathogen, Puccinia striiformis f. sp. tritici, causing yellow rust on wheat. Pathogen samples were collected between 1975 and 2002 in the UK and Denmark, and assayed for 14 individual avirulence/virulence alleles and up to 234 amplified fragment length polymorphism (AFLP) primer pairs producing approximately 17 000 AFLP fragments. The large number of fragments and a targeted sampling of isolates allowed a reconstruction of phylogenies in great detail, i.e. no homoplasy and a representation of sequential, evolutionary steps by pathogen samples. A recent, phenotypic loss of avirulence was observed at least once for loci corresponding to P. striiformis f. sp. tritici resistance Yr2, Yr3, Yr4, Yr7, Yr9, and Yr15, whereas Avr6 and Avr17 were lost independently in all three lineages, corresponding to 16 events of loss of avirulence (emergence of virulence). The opposite process, restoration of avirulence, was observed for Yr9 and Yr32. An interpretation of phenotypic changes within lineages as independent mutation events resulted in mutation frequencies from 1.4 × 10−6 to 4.1 × 10−6 per AFLP fragment (locus) per generation, whereas the effective rate by which a mutation from avirulence to virulence was established in the pathogen population, when subject to selection by host resistance genes, was approximately three orders of magnitude faster.
Polyandry in the wild: temporal changes in female mating frequency and sperm competition intensity in natural populations of the tettigoniid Requena verticalis
Authors: SIMMONS LEIGH W. , BEVERIDGE MAXINE , KENNINGTON W. JASON
Published: September 2007

Empirical tests of sexual selection theory generally utilize model systems under laboratory settings, and extend conclusions to evolutionary processes occurring in nature. The biological significance of laboratory findings will depend largely on the mating rates of females and patterns of paternity in natural populations, information on which is generally lacking. Here we use microsatellite markers to provide rare estimates of female mating rates and patterns of parentage in a species of tettigoniid, Requena verticalis, which has been used extensively to test theory on the evolution of male parental investment and its influence on the direction of sexual selection. We found that although the number of males having a genetic representation in the female's sperm stores was higher for females collected late in the breeding season than those collected early in the season, overall the female mating rate was lower than that expected from laboratory observations. Analysis of parentage of offspring produced by females at the end of the breeding season revealed that all males represented in the sperm stores fathered offspring, although paternity was biased away from that expected from random sperm utilization. The data show that the complete first male sperm precedence documented in laboratory studies of this species does not persist in natural populations. Our data provide a solid underpinning for conclusions drawn from laboratory studies of this species.
Coupling nucleotide hydrolysis to transcription activation performance in a bacterial enhancer binding protein
Authors: Joly Nicolas , Rappas Mathieu , Wigneshweraraj Siva R. , Zhang Xiaodong , Buck Martin
Published: September 2007

The bacterial enhancer binding proteins (bEBP) are members of the AAA+ protein family and have a highly conserved ‘DE’ Walker B motif thought to be involved in the catalytic function of the protein with an active role in nucleotide hydrolysis. Based on detailed structural data, we analysed the functionality of the conserved ‘DE’ Walker B motif of a bEBP model, phage shock protein F (PspF), to investigate the role of these residues in the σ54‐dependent transcription activation process. We established their role in the regulation of PspF self‐association and in the relay of the ATPase activity to the remodelling of an RNA polymerase·promoter complex (Eσ54·DNA). Specific substitutions of the conserved glutamate (E) allowed the identification of new functional ATP·bEBP·Eσ54 complexes which are stable and transcriptionally competent, providing a new tool to study the initial events of the σ54‐dependent transcription activation process. In addition, we show the importance of this glutamate residue in σ54·DNA conformation sensing, permitting the identification of new intermediate stages within the transcription activation pathway.
Systematic deletion analyses of the fla genes in the flagella operon identify several genes essential for proper assembly and function of flagella in the archaeon, Methanococcus maripaludis
Authors: Chaban Bonnie , Ng Sandy Y. M. , Kanbe Masaomi , Saltzman Ilana , Nimmo Graeme , Aizawa Shin‐Ichi , Jarrell Ken F.
Published: September 2007

The archaeal flagellum is a unique motility apparatus in the prokaryotic domain, distinct from the bacterial flagellum. Most of the currently recognized archaeal flagella‐associated genes fall into a single fla operon that contains the genes for the flagellin proteins (two or more genes designated as flaA or flaB), some variation of a set of conserved proteins of unknown function (flaC, flaD, flaE, flaF, flaG and flaH), an ATPase (flaI) and a membrane protein (flaJ). In addition, the flaD gene has been demonstrated to encode two proteins: a full‐length gene product and a truncated product derived from an alternate, internal start site. A systematic deletion approach was taken using the methanogen Methanococcus maripaludis to investigate the requirement and a possible role for these proposed flagella‐associated genes. Markerless in‐frame deletion strains were created for most of the genes in the M. maripaludis fla operon. In addition, a strain lacking the truncated FlaD protein [FlaD M(191)I] was also created. DNA sequencing and Southern blot analysis confirmed each mutant strain, and the integrity of the remaining operon was confirmed by immunoblot. With the exception of the ΔFlaB3 and FlaD M(191)I strains, all mutants were non‐motile by light microscopy and non‐flagellated by electron microscopy. A detailed examination of the ΔFlaB3 mutant flagella revealed that these structures had no hook region, while the FlaD M(191)I strain appeared identical to wild type. Each deletion strain was complemented, and motility and flagellation was restored. Collectively, these results demonstrate for first time that these fla operon genes are directly involved and critically required for proper archaeal flagella assembly and function.
Another piece of the puzzle of apoptotic cytochrome c release
Authors: Lawen Alfons
Published: September 2007

Involvement of the mitochondrial permeability transition pore (PTP) in apoptosis and PTP structure are highly controversial. In this issue of Molecular Microbiology, experiments based on yeast genetics analyse the roles of the three proteins commonly considered to form the PTP, i.e. porin, ADP/ATP carrier (ACC) and mitochondrial cyclophilin, on apoptosis‐like cell death. Whereas knocking out cyclophilin had no effect, the porin‐1 knockout yeast showed enhanced apoptosis, suggesting that porin‐1 has an antiapoptotic role. Loss of the ACC proteins afforded protection against some causes of death, but enhanced death induced by H2O2, suggesting a more complex role for the ACC proteins in regulating apoptosis‐like death in yeast.
YjbH is a novel negative effector of the disulphide stress regulator, Spx, in Bacillus subtilis
Authors: Larsson Jonas T. , Rogstam Annika , Von Wachenfeldt Claes
Published: September 2007

In the soil bacterium Bacillus subtilis Spx is a key regulator that controls expression, positively or negatively, of several genes in response to certain oxidative stresses that lead to the formation of unwanted disulphide bonds. Here we characterized the yjbH gene and show that it encodes a novel effector of Spx. The yjbH gene is part of the yjbIH operon that encodes a truncated haemoglobin (YjbI) and a predicted 34 kDa cytosolic protein of unknown function (YjbH). Deletion of yjbIH or yjbH has pleiotropic effects and affects growth, sporulation and competence development. Cells lacking yjbIH display a reduced sensitivity to the thiol oxidant diamide and show an apparent down‐ or upregulation of several transcripts that belong to the Spx regulon. Twenty‐two suppressor mutations that bypass the defects conferred by yjbH were isolated. These mutations were identified as six deletions, three nonsense and 11 missense substitutions in the spx gene. Real‐time quantitative reverse transcription polymerase chain reaction (qRT‐PCR) analysis showed that mutations in yjbIH or yjbH do not affect the level of spx transcription. The combined data from the present work show that strains lacking yjbIH or yjbH overproduce Spx under unperturbed growth. The elevated Spx concentration cannot be attributed to an increased spx expression but is likely to result from control at the post‐transcriptional level. YjbH is proposed to affect the cellular concentration of Spx by modulating proteolysis via the ClpXP protease.
A unique Mycobacterium ESX‐1 protein co‐secretes with CFP‐10/ESAT‐6 and is necessary for inhibiting phagosome maturation
Authors: Xu Junjie , Laine Olli , Masciocchi Mark , Manoranjan Joanna , Smith Jennifer , Du Shao Jun , Edwards Nathan , Zhu Xiaoping , Fenselau Catherine , Gao Lian‐Yong
Published: September 2007

The ESX‐1 secretion system plays a critical role in the virulence of Mycobacterium tuberculosis and M. marinum. To date, three proteins are known to be secreted by ESX‐1 and necessary for virulence, two of which are CFP‐10 and ESAT‐6. The ESX‐1 secretion and the virulence mechanisms are not well understood. In this study, we have examined the M. marinum secretomes and identified four proteins specific to ESX‐1. Two of those are CFP‐10 and ESAT‐6, and the other two are novel: MM1553 (homologous to Rv3483c) and Mh3881c (homologous to Rv3881c). We have shown that Mh3881c, CFP‐10 and ESAT‐6 are co‐dependent for secretion. Mh3881c is being cleaved at close to the C‐terminus during secretion, and the C‐terminal portion is critical to the co‐dependent secretion, the ESAT‐6 cellular levels, and interaction with ESAT‐6. The co‐dependent secretion is required for M. marinum intracellular growth in macrophages, where the Mh3881c C‐terminal portion plays a critical role. The role of the co‐dependent secretion in intracellular growth correlates with its role in inhibiting phagosome maturation. Both the secretion and the virulence defects of the Mh3881c mutant are complemented by Mh3881c or its M. tuberculosis homologue Rv3881c, suggesting that in M. tuberculosis, Rv3881c has similar functions.
The Hsp70 chaperone machines of Escherichia coli: a paradigm for the repartition of chaperone functions
Authors: Genevaux Pierre , Georgopoulos Costa , Kelley William L.
Published: September 2007

Molecular chaperones are highly conserved in all free‐living organisms. There are many types of chaperones, and most are conveniently grouped into families. Genome sequencing has revealed that many organisms contain multiple members of both the DnaK (Hsp70) family and their partner J‐domain protein (JDP) cochaperone, belonging to the DnaJ (Hsp40) family. Escherichia coli K‐12 encodes three Hsp70 genes and six JDP genes. The coexistence of these chaperones in the same cytosol suggests that certain chaperone–cochaperone interactions are permitted, and that chaperone tasks and their regulation have become specialized over the course of evolution. Extensive genetic and biochemical analyses have greatly expanded knowledge of chaperone tasking in this organism. In particular, recent advances in structure determination have led to significant insights of the underlying complexities and functional elegance of the Hsp70 chaperone machine.
Differential regulation of the transcriptional repressor NRG1 accounts for altered host‐cell interactions in Candida albicans and Candida dubliniensis
Authors: Moran Gary P. , MacCallum Donna M. , Spiering Martin J. , Coleman David C. , Sullivan Derek J.
Published: September 2007

Candida dubliniensis is genetically closely related to Candida albicans, but causes fewer infections in humans and exhibits reduced virulence and filamentation in animal models of infection. We investigated the role of the C. dubliniensis transcriptional repressor‐encoding gene CdNRG1 in regulating this phenotype. Deletion of both copies of CdNRG1 increased the formation of true hyphae by C. dubliniensis in response to serum, exogenous cAMP and CO2. In addition, deletion of CdNRG1 greatly enhanced filamentation and survival of C. dubliniensis in co‐culture with murine macrophages. In the reconstituted human oral epithelium infection model, the nrg1Δ mutant caused increased tissue damage relative to the wild‐type strain. However, deletion of CdNRG1 did not change the virulence of C. dubliniensis in the systemic mouse model of infection. The increased rate of hypha formation in C. albicans relative to C. dubliniensis in response to phagocytosis by macrophages and serum was associated with rapid downregulation of NRG1 expression in C. albicans. This study demonstrates that the reduced virulence and host cell damage elicited by C. dubliniensis may in part be due to the inability of this species to modulate NRG1 expression in response to the same environmental signals that promote filamentation in C. albicans.
A novel relaxase homologue is involved in chromosomal DNA processing for type IV secretion in Neisseria gonorrhoeae
Authors: Salgado‐Pabón Wilmara , Jain Samta , Turner Nicholas , Van Der Does Chris , Dillard Joseph P.
Published: September 2007

The Neisseria gonorrhoeae type IV secretion system secretes chromosomal DNA that acts in natural transformation. To examine the mechanism of DNA processing for secretion, we made mutations in the putative relaxase gene traI and used nucleases to characterize the secreted DNA. The nuclease experiments demonstrated that the secreted DNA is single‐stranded and blocked at the 5′ end. Mutation of traI identified Tyr93 as required for DNA secretion, while substitution of Tyr201 resulted in intermediate levels of DNA secretion. TraI exhibits features of relaxases, but also has features that are absent in previously characterized relaxases, including an HD phosphohydrolase domain and an N‐terminal hydrophobic region. The HD domain residue Asp120 was required for wild‐type levels of DNA secretion. Subcellular localization studies demonstrated that the TraI N‐terminal region promotes membrane interaction. We propose that Tyr93 initiates DNA processing and Tyr201 is required for termination or acts in DNA binding. Disruption of an inverted‐repeat sequence eliminated DNA secretion, suggesting that this sequence may serve as the origin of transfer for chromosomal DNA secretion. The TraI domain architecture, although not previously described, is present in 53 uncharacterized proteins, suggesting that the mechanism of TraI function is a widespread process for DNA donation.
Interaction of Bacteroides fragilis pLV22a relaxase and transfer DNA with Escherichia coli RP4‐TraG coupling protein
Authors: Thomas Johnson , Hecht David W.
Published: September 2007

Many Bacteroides transfer factors are mobilizable in Escherichia coli when coresident with the IncP conjugative plasmid RP4, but not F. To begin characterization and potential interaction between Bacteroides mobilizable transfer factors and the RP4 mating channel, both mutants and deletions of the DNA processing (dtr), mating pair formation (mpf) and traG coupling genes of RP4 were tested for mobilization of Bacteroides plasmid pLV22a. All 10 mpf but none of the four dtr genes were required for mobilization of pLV22a. The RP4 TraG coupling protein (CP) was also required for mobilization of pLV22a, but could be substituted by a C‐terminal deletion mutant of the F TraD CP. Potential interactions of the TraG CP with relaxase protein(s) and transfer DNA of both RP4 and pLV22a were assessed. Overlay assays identified productive interactions between TraG and the relaxase proteins of both MbpB and TraI from pLV22a and RP4 respectively. The Agrobacterium Transfer‐ImmunoPrecipitation (TrIP) assay also identified an interaction between TraG and both RP4 and pLV22a transfer DNA. Thus, mobilization of the Bacteroides pLV22a in E. coli utilizes both RP4 Mpf and CP functions including an interaction between the relaxosome and the RP4 CP similar to that of cognate RP4 plasmid.
Feeding by the tick, Ixodes scapularis, causes CD4+ T cells responding to cognate antigen to develop the capacity to express IL‐4
Authors: MÜLLER‐DOBLIES U. U. , MAXWELL S. S. , BOPPANA V. D. , MIHALYO M. A. , MCSORLEY S. J. , VELLA A. T. , ADLER A. J. , WIKEL S. K.
Published: September 2007

Effects of tick feeding on an early antigen‐specific T cell response were studied by monitoring a clonotypic population of adoptively transferred T cell receptor (TCR) transgenic CD4 cells responding to a tick‐associated antigen. When recipient mice were infested with pathogen‐free Ixodes scapularis nymphs several days prior to T cell transfer and intradermal injection of soluble cognate antigen at the feeding site, the clonotypic CD4 cells gained the ability to express the Th2 effector cytokine IL‐4. Notably, this effect was not only observed in BALB/c mice predisposed towards developing Th2 responses but also in B10.D2 mice predisposed towards Th1 responsiveness. Furthermore, tick feeding was able to superimpose IL‐4 expression potential onto a strong Th1 response (indicated by robust IFN‐γ expression potential) elicited by immunization with a vaccinia virus expressing the cognate antigen. The magnitude to which tick feeding was able to programme IL‐4 expression potential in CD4 cells was partially reduced in mice that had been previously exposed to pathogen‐free tick nymphs 6 weeks earlier, as well as when the nymphs were infected with Borrelia burgdorferi. Intradermal injection of salivary gland extract programmed IL‐4 expression potential similar to that of tick infestation, suggesting that IL‐4 programming activity is contained within tick saliva.
Wound healing response is a major contributor to the severity of cutaneous leishmaniasis in the ear model of infection
Authors: BALDWIN T. , SAKTHIANANDESWAREN A. , CURTIS J. M. , KUMAR B. , SMYTH G. K. , FOOTE S. J. , HANDMAN E.
Published: September 2007

In the conventional mouse model for cutaneous leishmaniasis involving infection with stationary phase Leishmania major promastigotes at the base of the tail, mice congenic for leishmaniasis resistance loci designated lmr1,2,3 cured their lesions more rapidly and laid down more ordered collagen fibres than the susceptible parental BALB/c mice, while the opposite was the case for the congenic mice carrying the susceptibility loci on the resistant C57BL/6 background. In that model, we showed that wound healing and not T cell responses played a major role in determining the resolution of skin infection. Here, we show a similar disease phenotype in the mouse model that mimics more closely the situation in humans, that is, strictly intradermal infection in the ear pinna with small numbers of metacyclic promastigotes. The data show that at the site of infection the innate and adaptive immune responses act in concert to clear parasites, and induce tissue repair and wound healing. Importantly, the data show that the host responses controlled by the lmr loci, which act locally to control infection in the skin, are distinct from the host responses operating systemically in the draining lymph node.
Inhibition of groups 1 and 2 CD1 molecules on human dendritic cells by Leishmania species
Authors: DONOVAN M. J. , JAYAKUMAR A. , McDOWELL M. A.
Published: September 2007

Dendritic cells are potent immune‐activating cells, which traditionally are thought of as presenters of protein antigen to lymphocytes to initiate an immune response. Recently, another mechanism of immune surveillance, the detection of lipid antigens, has been found to be mediated by the nonpolymorphic family of CD1 molecules. There are two different CD1 families, Group 1 consisting of CD1a, CD1b and CD1c, and Group 2 consisting only of CD1d. Leishmania donovani‐infected dendritic cells have previously been demonstrated to exhibit decreased surface levels of Group 1 CD1 molecules and are no longer able to initiate a CD1b‐restricted T cell response. In contrast to L. donovani, which disseminates to the visceral organs, L. major remains localized, forming a cutaneous lesion. We investigate here whether L. major, the aetiological agent of cutaneous leishmaniasis, also inhibits CD1 expression. We demonstrate that infection of human monocyte‐derived dendritic cells with either L. major or L. donovani results in transcriptional down‐regulation of both Groups 1 and 2 CD1 molecules. Furthermore, infection of monocytes during differentiation results in a cell phenotype similar to undifferentiated monocytes. Finally, we demonstrate that this down‐regulation is not mediated by lipophosphoglycan or other phosphoglycans.
Immunogenicity in rhesus of the Plasmodium vivax mosquito stage antigen Pvs25H with Alhydrogel and Montanide ISA 720
Authors: SAUL A. , HENSMANN M. , SATTABONGKOT J. , COLLINS W. E. , BARNWELL J. W. , LANGERMANS J. A. M. , WU Y. , LONG C. A. , DUBOVSKY F. , THOMAS A. W.
Published: September 2007

Pvs25 is an ookinete surface protein from Plasmodium vivax that is the target of transmission‐blocking antibodies. Two immunogenicity trials in rhesus monkeys with a recombinant form of the protein, Pvs25H, were undertaken. Monkeys were vaccinated with Pvs25H adsorbed to Alhydrogel or emulsified in Montanide ISA 720 at 0, 4 and 27 weeks (study 1) or in Montanide ISA 720 at 0 and 18 weeks (study 2) with 1·5 or 15 µg Pvs25H in 0·1 or 0·5 mL of emulsion (four combinations). Immunogenicity was assessed by ELISA and by membrane‐feeding experiments using P. vivax‐infected blood from human volunteers (studies 1 and 2) or from chimpanzees (study 1). Both vaccine trials generated antibodies that blocked transmission of P. vivax to mosquitoes. Antibody titres and transmission blocking were higher with Montanide ISA 720 than with Alhydrogel in the first trial and with the 15 µg Pvs25H/0·5 mL ISA 720 combination in the second trial.
Parasite‐specific IL‐4 responses inAscaris suumandTrichuris suis‐infected pigs evaluated by ELISPOT
Authors: STEENHARD N. R. , KRINGEL H. , ROEPSTORFF A. , THAMSBORG S. M. , JUNGERSEN G.
Published: September 2007

The objective of the present study was to develop an ELISPOT method to measure parasite‐specific IL‐4 producing cells during experimentalAscaris suumandTrichuris suisinfections in pigs. In many experimental settings it is useful to be able to measure changes in specifically induced cytokines over time at post‐mRNA level; in particular, specific measurement of IL‐4 is important for studies on nematodes due to the key function of IL‐4 in driving the Th2 response. Two separate experiments were carried out, one withA. suumand other withT. suisinfection in which we were able to measure statistically significant increases in specific IL‐4 production in peripheral blood mononuclear cells over time in parallel to an increase in blood eosinophils. Furthermore, IL‐4 was measured in the colon lymph node ofT. suis‐infected pigs. Egg excretion and worm burdens at necropsy were measured. The ELISPOT method is a valuable tool for future experimental settings as it enables repeated and parasite‐specific measurement of IL‐4 at protein level when investigating, for example, immunomodulatory properties of helminths. Furthermore, the method could be used to identify specific parasite antigens inducing IL‐4 production.