bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical significance of neutrophil CD177 mRNA expression in Ph‐negative chronic myeloproliferative disorders
Authors: Passamonti Francesco , Pietra Daniela , Malabarba Lucia , Rumi Elisa , Porta Matteo Giovanni Della , Malcovati Luca , Bonfichi Maurizio , Pascutto Cristiana , Lazzarino Mario , Cazzola Mario
Published: August 2004

The PRV‐1 gene has been proposed as a marker of polycythaemia vera (PV). PRV‐1 and NB1 are alleles of the polymorphic gene CD177, which belongs to the Ly‐6/uPAR superfamily, and their coding regions differ at only four nucleotides. We studied neutrophil CD177 mRNA levels in normal subjects and in 235 patients with Ph‐negative chronic myeloproliferative disorders (CMD), including PV, essential thrombocythaemia and myelofibrosis with myeloid metaplasia. Additional disease states were investigated for comparison. Highly variable neutrophil CD177 mRNA levels were observed in normal individuals. Neutrophils isolated from the bone marrow, or from peripheral blood following granulocyte colony‐stimulating factor administration showed markedly higher CD177 expression than circulating granulocytes on steady state. Increased neutrophil CD177 mRNA levels were detected in all CMD. Elevated values were also found in reactive conditions and in disorders such as chronic myeloid leukaemia and myelodysplastic syndromes. In the differential diagnosis between PV and secondary erythrocytosis, the assay sensitivity was 68% while its specificity was 60%. These findings indicate that an elevated neutrophil CD177 mRNA level is not a specific marker for the diagnosis of PV nor for that of CMD. From a clinical viewpoint, neutrophil CD177 mRNA overexpression is rather a marker of abnormal neutrophil production and/or release in patients with CMD.
Human herpesvirus 6 antigenaemia in allogeneic stem cell transplant recipients: impact on clinical course and association with other beta‐herpesviruses
Authors: Volin Liisa , Lautenschlager Irmeli , Juvonen Eeva , Nihtinen Anne , Anttila Veli‐Jukka , Ruutu Tapani
Published: August 2004

Human herpesvirus 6 (HHV‐6) antigenaemia was prospectively studied in 58 adult allogeneic stem cell transplant (SCT) recipients. Altogether 42 of 58 recipients (72%) demonstrated HHV‐6 specific antigens in peripheral blood mononuclear cells after SCT, 22 of 36 (61%) when the donor was a sibling and 20 of 22 (91%) when the donor was unrelated. The cumulative incidence of HHV‐6A, HHV‐6B, HHV‐7, and cytomegalovirus antigenaemia during the first 6 months after SCT was 33%, 62%, 44% and 63% respectively. The median day of the onset of each antigenaemia was +24, +4, +59, and +46 after SCT respectively. There were no clinical findings related to HHV‐6A and HHV‐7 antigenaemias. A rash was diagnosed in 10 of 38 (26%) HHV‐6B antigenaemia positive patients during the first month after SCT compared with one of 20 (5%) HHV‐6B negative patients. Of the HHV‐6B antigenaemia cases, six of 10 rashes were treated as acute graft‐versus‐host disease (GVHD) and four of 10 were considered to be of a viral origin. Fifteen patients had acute GVHD diagnosed. Acute GVHD manifested statistically significantly (P = 0·034) earlier in the nine patients with HHV‐6B antigenaemia compared with the six patients who were HHV‐6B negative.
Suppressor of cytokine signalling‐1 gene silencing in acute myeloid leukaemia and human haematopoietic cell lines
Authors: Watanabe Dai , Ezoe Sachiko , Fujimoto Minoru , Kimura Akihiro , Saito Yoshiyuki , Nagai Hisaki , Tachibana Isao , Matsumura Itaru , Tanaka Toshio , Kanegane Hirokazu , Miyawaki Toshio , Emi Mitsuru , Kanakura Yuzuru , Kawase Ichiro , Naka Tetsuji , Kishimoto Tadamitsu
Published: August 2004

The aim of this study was to investigate whether the suppressor of cytokine signalling (SOCS)‐1 can act as a tumour suppressor when functioning as a negative regulator of the Janus family tyrosine kinases (JAKs), which have been reported to play important roles in leukaemogenesis. For this purpose, we carried out molecular analysis of the SOCS‐1 gene in human acute myeloid leukaemia (AML) and human haematopoietic cell lines. Sequencing alterations in the coding region were found in two of 90 primary AML samples and one of 17 cell lines. Hypermethylation of the SOCS‐1 gene was also observed in 72% of primary cases and 52% of cell lines and aberrant methylation strongly correlated with reduced expression. Transfection of SOCS‐1 into Jurkat cells harbouring the mutation and methylation suppressed cell growth at a low serum concentration. These findings indicate that SOCS‐1 is frequently silenced in haematopoietic malignancies, mainly as a result of hypermethylation, and suggest that SOCS‐1 may be able to function as a tumour suppressor.
Kinetic of regulatory CD25high and activated CD134+ (OX40) T lymphocytes during acute and chronic graft‐versus‐host disease after allogeneic bone marrow transplantation
Authors: Sanchez Joaquin , Casaño Javier , Alvarez Miguel A. , Roman‐Gomez Jose , Martin Carmen , Martinez Francisco , Gomez Pedro , Serrano Josefina , Herrera Concepcion , Torres Antonio
Published: August 2004

Graft‐versus‐host disease (GVHD) is still a major complication after allogeneic stem cell transplantation. In murine models, freshly isolated or ex vivo expanded CD4+CD25high regulatory T cells (Treg) are able to ameliorate GVHD while maintaining graft‐versus‐leukaemia reactions. However, in the human setting, prospective studies of this population and its interaction with activated non‐regulatory CD134+ (OX40) lymphocytes during post‐transplant follow‐up are lacking. In this study, we prospectively quantified CD4+CD25high and activated CD134+ lymphocytes in 119 peripheral blood samples from 35 consecutive patients who underwent allogeneic bone marrow transplantation (BMT). Fifty‐five samples obtained less than 100 d after allogeneic BMT, were not statistically different regarding CD4+CD25high Treg or CD134+ lymphocytes compared with those obtained from patients with (n = 35) or without (n = 20) acute GVHD. Chronic GVHD was associated with a small, but not statistically significant, increase in the number of Treg (9·9 vs. 6·7 × 106/L). However, the CD134/CD25high ratio was significantly higher during chronic GVHD (cGHVD) when compared with either patients without cGVHD (67·7 ± 40·3 vs. 4·0 ± 0·9, P < 0·01) or cGVHD after treatment (67·7 ± 40·3 vs. 3·7 ± 0·8, P < 0·01). Our findings suggest that the suppressive activity of CD4+CD25high Treg could be abrogated in vivo during cGVHD by CD134 expression in a much higher number of activated donor T lymphocytes. In addition to CD4+CD25highex vivo expansion protocols, OX40 blocking might be crucial to optimize the use of Treg to prevent GVHD.
Detection of serine 473 phosphorylated Akt in acute myeloid leukaemia blasts by flow cytometry
Authors: Tazzari Pier Luigi , Cappellini Alessandra , Grafone Tiziana , Mantovani Irina , Ricci Francesca , Billi Anna Maria , Ottaviani Emanuela , Conte Roberto , Martinelli Giovanni , Martelli Alberto M.
Published: August 2004

The phosphoinositide 3‐kinase/Akt signalling pathway is a recently recognized important parameter in the prognosis and the response to treatment of acute myeloid leukaemia (AML). Akt kinase is activated by phosphorylation on Thr 308 and Ser 473. Active Akt promotes cell growth and survival to apoptotic insults. Thus, it seems important to evaluate Akt phosphorylation in AML blasts. This work aimed to establish whether it was possible to detect Akt phosphorylation on Ser 473 of AML blasts by means of flow cytometry. High levels of Akt activity and phosphorylation were detected in 13 of 15 cases of AML. Flow cytometric analysis revealed similar patterns of Ser 473 expression as was observed with Akt kinase activity and Western blot analysis of Thr 308 and Ser 473 phosphorylation. Double immunostaining enabled the simultaneous flow cytometric detection of an AML‐associated antigen (CD33) and Ser 473 phosphorylated Akt in leukaemic blast populations. Our results indicate that flow cytometry enabled the rapid and quantitative assessment of Ser 473 phosphorylated Akt of AML blasts that, when used in combination with cell surface staining, can provide more accurate phenotyping of AML blasts.
Patient interleukin‐18 GCG haplotype associates with improved survival and decreased transplant‐related mortality after unrelated‐donor bone marrow transplantation
Authors: Cardoso Sandra M. P. , DeFor Todd E. , Tilley Louise A. , Bidwell Jeffrey L. , Weisdorf Daniel J. , MacMillan Margaret L.
Published: August 2004

Interleukin‐18 (IL‐18), a proinflammatory cytokine, is elevated in patients with acute graft‐versus‐host disease (aGVHD). IL‐18 induces Th1 differentiation and cytotoxic T‐lymphocyte function, both of which have been implicated in the pathogenesis of aGVHD. However, recent studies have shown that neutralization of IL‐18 by antibodies leads to an increased risk of aGVHD‐related mortality while administration of IL‐18 significantly improved survival. We have genotyped a cohort of 157 patient/donor pairs undergoing unrelated donor bone marrow transplantation (BMT) for three polymorphisms recently identified in the promoter of the IL‐18 gene: G‐137C, C‐607A and G‐656T. Usingphasesoftware, three main haplotypes were reconstructed: GCG, CAT and GAT. We found no association between the occurrence of aGVHD and patient/donor haplotypes. The presence of the GCG haplotype in patients was associated with significantly decreased risk of transplant‐related mortality at 100 d (23% in patients with GCG vs. 48% in patients without GCG,P < 0·01) and at 1 year (36% vs. 65%,P < 0·01). The presence of the GCG haplotype in patients was also associated with improved survival (57% vs. 32%,P < 0·01). Cox regression analysis showed that the presence of the GCG haplotype was associated with a twofold increased probability of survival. These data suggest that the IL‐18 promoter GCG haplotype may influence survival after unrelated donor BMT without altering the risk of aGVHD.
Unrelated cord blood transplantation after myeloablative conditioning in patients over the age of 45 years
Authors: Ooi Jun , Iseki Tohru , Takahashi Satoshi , Tomonari Akira , Takasugi Kashiya , Uchiyama Michihiro , Konuma Takaaki , Futami Muneyoshi , Nomura Akiko , Nakayama Shin , Soda Yasushi , Ohno Nobuhiro , Nagamura Fumitaka , Uchimaru Kaoru , Tojo Arinobu , Tani Kenzaburo , Asano Shigetaka
Published: August 2004

We report the results of unrelated cord blood transplantation (CBT) after myeloablative conditioning in 21 patients over the age of 45 years. Among the patients the median age was 48 years (range, 45–53 years), the median weight was 58·6 kg (range, 43·6–76·2 kg) and the median number of cryopreserved nucleated cells was 2·45 × 107/kg (range, 1·63–3·71 × 107/kg). Nineteen patients had myeloid reconstitution and the median time to more than 0·5 × 109/l absolute neutrophil count was 22 d. A self‐sustained platelet count more than 50 × 109/l was achieved in 17 patients at a median time of 49 d. Acute graft‐versus‐host disease (GVHD) above grade II occurred in 7 of 19 evaluable patients and chronic GVHD occurred in 14 of 16 evaluable patients. Among 14 chronic GVHD patients, in seven patients the disease was extensive. Fifteen patients were alive and free of disease at between 217 and 1798 d after transplantation. With a median follow‐up of 847 d, the probability of disease‐free survival at 2 years was 71·4%. These results suggest that patients over 45 years of age without suitable related or unrelated bone marrow donors should be considered as candidates for CBT.
Geographical zonation in the Neotropics of tree species characteristic of the Paraguay‐Paraná Basin
Authors: Spichiger Rodolphe , Calenge Clément , Bise Bastian
Published: August 2004

Aim  This paper uses data from the literature (monographs and taxonomic reviews) to investigate the geographical zonation in South America of 32 common tree species encountered in Paraguay. The actual spatial distribution of the species is then used to provide clues on the plant communities present in the past, and especially during the Wisconsinan age (Last major ice age: 80,000–10,000 bp). The floristic relatedness between the Paraguayan flora and the neighbouring floras is also investigated.

Methods  The main vegetation patterns were highlighted using a Discriminant Analysis of the Eigenvectors of Neighbourhood Operator to emphasize the geographical zonation in South America of 32 tree species predominant in the Paraguay‐Paraná Basin.

Results  Three main vegetation patterns were emphasized: the Chaco, the Paraná‐São Francisco gradient and the Peri‐Amazonian gradient. The Chaco is well defined. The Peri‐Amazonian gradient is characterized by a continuous change in species from the Colombian pole towards the São Francisco pole. The São Francisco gradient shows a continuous change from the São Francisco pole towards the Paraná pole. Some of the species are monocentric (related to only one dispersal centre), whereas others are polycentric (distributed in several poles).

Main conclusions  The Chaquean xeromorphic forests are considered as a climax, with a stable composition resulting of the saline soil conditions. The Chaco acts as an edaphic barrier to many species, although the migration of some non‐Chaquean species is made possible by the net of gallery‐forests and low montane forests between the Río Paraguay and the Andean Piedmont. The Cerrado and the Chaco can be considered as a barrier limiting the expansion of the Paranean forest. This latter formation is a semi‐deciduous forest made of a mix of monocentric and polycentric elements. The numerous species found both in Colombia and in Paraná indicate that bridges exist, or have existed, between these two poles. The persistence in the Paraguay‐Paraná Basin of a moist forest made of the Paranean assemblages highlighted in this study confirms the hypothesis of moister and cooler phases than today.
Diversity and biogeography of vascular epiphytes in Western Amazonia, Yasuní, Ecuador
Authors: Kreft Holger , Köster Nils , Küper Wolfgang , Nieder Jürgen , Barthlott Wilhelm
Published: August 2004

Aim  Although vascular epiphytes are important components of species richness and complexity of Neotropical forests, vascular epiphytes are under‐represented in large scale biogeographical analyses. We studied the diversity, biogeography and floristic relationships of the epiphytic flora of the Yasuní region (Western Amazonia) in a Neotropical context, with special emphasis on the influence of the Andean flora on floristic composition and diversity of surrounding lowland forests.

Location  Western Amazonian lowland rainforest, Tiputini Biodiversity Station (0°38′ S 76°09′ W, 230 m a.s.l., 650 ha), Yasuní National Park, Ecuador.

Methods  We compared the vascular epiphyte flora of Yasuní with 16 published Neotropical epiphyte inventories. Secondly, based on a floristic database with records of more than 70,000 specimens of vascular epiphytes from the Neotropics the elevational composition of eight selected inventories was analysed in detail.

Results  The vascular epiphyte flora of Yasuní is characterized by a very high species richness (313 spp.). A moderate portion of species is endemic to the Upper Napo region (c. 10%). However, this figure is much higher than previous analyses primarily based on woody species suggested. Geographical ranges of these species match with a proposed Pleistocene forest refuge. Compared with Northern and Central Amazonian sites, Western Amazonian epiphyte communities are characterized by a higher portion of montane and submontane species. Species richness of vascular epiphytes at the sites was correlated with the amount of rainfall, which is negatively correlated with the number of dry months.

Main conclusion  Recent and historic patterns of rainfall are the driving forces behind diversity and floristic composition of vascular epiphytes in Western Amazonia: high annual rainfall in combination with low seasonality provides suitable conditions to harbour high species richness. The proximity to the Andes, the most important centre of speciation for most Neotropical epiphytic taxa, in combination with the climatic setting has allowed a continuous supply of species richness to the region. At least for epiphytes, the borderline between the Andean and Amazonian flora is much hazier than previously thought. Moreover, the comparatively moist climate in Western Amazonia during the Pleistocene has probably led to fewer extinctions and/or more speciation than in more affected surrounding lowlands.
Large‐scale diversity patterns of vascular epiphytes in Neotropical montane rain forests
Authors: Küper W. , Kreft H. , Nieder J. , Köster N. , Barthlott W.
Published: August 2004

Aim  Epiphytes contribute up to 30% to the number of vascular plant species in certain global biodiversity hotspots, e.g. the Ecuadorian Andes. However, their large scale diversity patterns are still discussed on the base of results from a few, local epiphyte inventories. Consequently, explanatory models on epiphyte diversity concentrate on the impact of local climate on small scale epiphyte species richness. Our aim was to analyse large scale elevational patterns of epiphyte diversity integrating data from different geographic scales.

Location  Tropical America, with special emphasis on the Ecuadorian Andes.

Methods  Our study is based on two data sources. First, we analysed the elevational patterns of epiphyte diversity based on the Catalogue of the Vascular Plants of Ecuador and the Libro Rojo de las Plantas Endèmicas del Ecuador. Secondly, the floristic turnover between the epiphyte inventories of seven montane and four lowland study sites in the Neotropics was analysed.

Results  The floristic turnover between Neotropical montane epiphyte floras is higher than the one between lowland epiphyte floras. Montane study sites located only a few kilometres apart from each other show considerable differences in their epiphyte species inventories. Irrespectively of their similar dispersal mode, the floristic turnover is much higher for orchids than for Pteridophyta. The Orchidaceae are the species richest group in all of the examined 11 Neotropical epiphyte floras. At the larger scale of the Ecuadorian Flora, c. 50% of the species in the elevational zone with maximum epiphyte diversity (between 1000 and 1500 m) are orchids. Elevational patterns of epiphyte diversity strongly reflect patterns of Orchidaceae.

Main conclusions  Our results support the observation of a ‘mid‐elevation bulge’ of epiphyte diversity by Gentry and Dodson. It has been frequently shown that the high humidity in mid‐elevations is suitable to maintan a high epiphyte species richness. Our findings show that in addition, large scale epiphyte diversity in montane rain forest is increased by the high floristic turnover at local and regional scale. Based on the importance of Orchidaceae for epiphyte diversity, we discuss that speciation processes corresponding to the highly diverse environment are a driving force for endemism, floristic heterogeneity and consequently for large scale epiphyte species richness in montane forests.
Forest progression modes in littoral Congo, Central Atlantic Africa
Authors: Favier Charly , De Namur Christian , Dubois Marc‐Antoine
Published: August 2004

Aim  To understand the persistence of a forest–savanna mosaic in places where rainfall data suggest that forest take‐over should take place. To study the various modes of forest encroachment, and the role of human activities to hamper it.

Location  Data were collected on several forest–savanna ecotones in the coastal region of the Republic of Congo. The sites were chosen to illustrate the differing principal modes of forest expansion, corresponding to different levels of anthropic pressure.

Methods  The study sites were situated on five transects perpendicular to the ecotone (total sampled area: 1.7 ha) and 10 forest clumps in savanna (with diameters from 3 to 20 m). Along the transects botanical identification, diameter measurement and cartography were performed, while leaf area index was measured at a high resolution (every metre) along two of them. Collected data were analysed using a continuous quantification approach, which is much more useful than classical quadrat analysis. Time calibration of progression rates was performed using a simple model of the growth of the characteristic pioneer species, Aucoumea klaineana.

Results  The two main different modes are reflected in different successional patterns. The edge diffusion is slow (its rate is evaluated to c. 1 m year−1) and is characterized by a progressive increase in large‐diameter tree density and shade‐tolerant tree density away from the ecotone. Conversely, savanna to forest phase transition by coalescence of clumps exhibits high tree density remnants distributed in established forest. The composition of these remnants is compatible with that of the forest clumps in savannas.

Main conclusions  Three functional groups of pioneer trees are distinguished: some occupy the edge (edge pioneer), others establish clumps of forest in savanna (clump pioneers) and the longer‐living A. klaineana ensures the transition to ‘mature’ forest. The two different observed patterns (linear edge progression and clump coalescence) can be understood with the use of a model of forest–savanna dynamics, ‘FORSAT’. The two control parameters are the annual rainfall and the frequency of man‐made fires in each savanna. In particular, an increase in the fire frequency can lead to a shift from the coalescence regime to the edge progression one.
The Holocene spread of Picea abies (L.) Karst. in Fennoscandia and adjacent areas
Authors: Giesecke Thomas , Bennett K. D.
Published: August 2004

Aim  The Holocene spread of Picea abies in Fennoscandia is well established from many sites and thus provides an opportunity for detailed study of the dynamics of tree spread and population expansion. Early‐ and mid‐Holocene macrofossil evidence for presence of P. abies in Fennoscandia has questioned traditional interpretations of the timing and direction of its spread. This paper aims to determine when, from where and by which pathways P. abies spread into Fennoscandia. Understanding the character and dynamics of this spread may give insight into the general understanding of Holocene tree spread.

Location  The north‐western distribution of P. abies in Europe, including Norway, Sweden, Finland, Estonia, Latvia, Lithuania, north‐western Russia, parts of Byelorussia and Poland.

Methods  Holocene pollen diagrams with independent dating control were collected from this region. The timing of the onset of the continuous curve, the timing of the rise of the curve, the first appearance of frequencies of 1%, 3%, 5%, and 10%, as well as timing and the maximum amount of P. abies pollen, was obtained from these pollen diagrams. A GIS analysis was used to display the data and interpolate ages over the area under investigation.

Results  Maps are presented showing a clear ESE to WNW trend in the spread of P. abies for all characters interpolated. The timing of the rise of the curve was difficult to use as sites east of the Baltic have slowly rising P. abies frequencies while the western sites often show a rapid rise.

Main conclusions  The spread of P. abies in Fennoscandia and adjacent areas can be separated into two phases: (i) A rapid early Holocene spread out of Byelorussia and northern Russia at low population density giving rise to small outpost populations, possibly as far west as the Scandes Mountains. (ii) A mid‐ to late Holocene front‐like spread at high population densities moving from east to west into the Baltic Republics and Finland, into northern Scandinavia and then moving south and west towards its present‐day distributional limits.
A small, cysteine‐rich protein secreted by Fusarium oxysporum during colonization of xylem vessels is required for I‐3‐mediated resistance in tomato
Authors: Rep Martijn , Van Der Does H. Charlotte , Meijer Michiel , Van Wijk Ringo , Houterman Petra M. , Dekker Henk L. , De Koster Chris G. , Cornelissen Ben J. C.
Published: August 2004

A 12 kDa cysteine‐rich protein is secreted by Fusarium oxysporum f. sp. lycopersici during colonization of tomato xylem vessels. Peptide sequences obtained with mass spectrometry allowed identification of the coding sequence. The gene encodes a 32 kDa protein, designated Six1 for secreted in xylem 1. The central part of Six1 corresponds to the 12 kDa protein found in xylem sap of infected plants. A mutant that had gained virulence on a tomato line with the I‐3 resistance gene was found to have lost the SIX1 gene along with neighbouring sequences. Transformation of this mutant with SIX1 restored avirulence on the I‐3 line. Conversely, deletion of the SIX1 gene in a wild‐type strain results in breaking of I‐3‐mediated resistance. These results suggest that I‐3‐mediated resistance is based on recognition of Six1 secreted in xylem vessels.
Regulatory roles for the homeodomain and C2H2 zinc finger regions of Cryptococcus neoformans Ste12αp
Authors: Chang Yun C. , L. C. Wright  , Tscharke R. L. , Sorrell T. C. , Wilson C. F. , Kwon‐Chung K. J.
Published: August 2004

The STE12α gene of Cryptococcus neoformans encodes a protein containing both homeodomain and zinc finger regions. As homeodomains and zinc finger regions are important domains for the function of many transcription factors, we used site‐specific mutagenesis to delineate the roles of these two domains. The homeodomain and zinc finger regions are each important for the function of Ste12αp. DNA binding ability, mating frequency, and haploid fruiting capability were reduced in strains with mutations in the homeodomain, whereas virulence and capsule size in the mouse brain were increased. In contrast, mutations in the zinc fingers region resulted in decreased virulence, reduced capsule size in the mouse brain and decreased gene expression of capsule associated genes. In addition, phospholipase activity was increased in the zinc finger mutants. Taken together, most of the phenotypes previously observed in the ste12α deletion strains were reproduced in these two types of mutants. However, unlike mutations in the homeodomain/zinc finger region, complete deletion of STE12α caused a severe reduction in virulence and a decrease in phospholipase activity. These data suggest that region(s) other than the homeodomain and zinc finger regions of Ste12αp contribute to the variable influences on the different phenotypes observed in C. neoformans.
Methionine sulphoxide reductase is an important antioxidant enzyme in the gastric pathogen Helicobacter pylori
Authors: Alamuri Praveen , Maier Robert J.
Published: August 2004

The ability of Helicobacter pylori to colonize the stomach requires that it combat oxidative stress responses imposed by the host. The role of methionine sulfoxide reductase (Msr), a methionine repair enzyme, in H. pylori stress resistance was evaluated by a mutant analysis approach. An msr mutant strain lacked immunologically detectable sulphoxide reductase protein and also showed no enzyme activity when provided with oxidized methionines as substrates. The mutant strain showed diminished growth compared to the parent strain in the presence of chemical oxidants, and showed rapid viability loss when exposed to oxidizing conditions. The stress resistance and enzyme activity could be recovered by complementing the mutant with a functional copy of the msr gene. Upon fractionation of parent strain and the complemented mutant cells into membranes and cytoplasmic proteins, most of the immunologically detectable Msr was localized to the membrane, and this fraction contained all of the Msr activity. Qualitative detection of the whole cell protein pattern using 2,4‐dinitro phenyl hydrazine (DNPH) showed a far greater number of oxidized protein species in the mutant than in the parent strain when the cells were subjected to oxygen, peroxide or s‐nitrosoglutathione (GSNO) induced stress. Importantly, no oxidized proteins were discerned in either strain upon incubation in anaerobic conditions. A mutant strain that synthesized a truncated Msr (corresponding to the MsrA domain) was slightly more resistant to oxidative stress than the msr strain. Mouse colonization studies showed Msr is an important colonization factor, especially for effective longer‐term (14 and 21 days) colonization. Complementation of the mutant msr strain by chromosomal insertion of a functional gene restored mouse colonization ability.
Htd2p/Yhr067p is a yeast 3‐hydroxyacyl‐ACP dehydratase essential for mitochondrial function and morphology
Authors: Kastaniotis Alexander J. , Autio Kaija J. , Sormunen Raija T. , Hiltunen J. Kalervo
Published: August 2004

Among the recently recognized aspects of mitochondrial functions, in yeast as well as humans, is their ability to synthesize fatty acids in a malonyl‐CoA dependent manner. We describe here the identification of the 3‐hydroxyacyl‐ACP dehydratase involved in mitochondrial fatty acid synthesis. A colony‐colour‐sectoring screen was applied in Saccharomyces cerevisiae in a search for mutants that, when grown on a non‐fermentable carbon source, were unable to lose a plasmid that carried a chimeric construct coding for mitochondrially localized bacterial analogue. Our mutants, which are respiratory deficient, lack cytochromes and display abnormal mitochondrial morphology,  were  found  to  have  a lesion in the yeast YHR067w/RMD12 gene. The Yhr067p is predicted to be a member of the thioesterase/thioester dehydratase‐isomerase superfamily enzymes. Hydratase 2 activity in mitochondrial extracts from cells overexpressing YHR067w was increased. These overexpressing cells also display a striking mitochondrial enlargement phenotype. We conclude that YHR067w encodes a novel mitochondrial 3‐hydroxyacyl‐thioester dehydratase 2 and suggest renaming it HTD2. The mitochondrial phenotypes of the null and overexpression mutants suggest a crucial role of YHR067w in maintenance of mitochondrial respiratory competence and morphology in yeast.
MexAB‐OprM hyperexpression in NalC‐type multidrug‐resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720‐PA3719
Authors: Cao Lily , Srikumar Ramakrishnan , Poole Keith
Published: August 2004

MexAB‐OprM is a multidrug efflux system that contributes to intrinsic and acquired multidrug resistance in Pseudomonas aeruginosa, the latter as a result of mutational hyperexpression of the mexAB‐oprM operon. While efflux gene hyperexpression typically results from mutations in the linked mexR repressor gene, it also occurs independently of mexR mutations in so‐called nalC mutants that demonstrate more modest mexAB‐oprM expression and, thus, more modest multidrug resistance than do mexR strains. Using a transposon insertion mutagenesis approach, nalC mutant strains were selected and the disrupted gene, PA3721, identified. Amplification and sequencing of this gene from previously isolated spontaneous nalC mutants revealed the presence of mutations in all instances and as such, PA3721 has been renamed nalC. PA3721 (nalC) encodes a probable repressor of the TetR/AcrR family and occurs upstream of an apparent two‐gene operon, PA3720–PA3719, whose expression was negatively regulated by PA3721. Thus, PA3720‐PA3719 was hyperexpressed in transposon insertion and spontaneous nalC mutants. The loss of PA3719 but not of PA3720 expression in a spontaneous nalC mutant reduced MexAB‐OprM expression to wild‐type levels and compromised multidrug resistance, an indication that hyperexpression of PA3719 only was necessary for the nalC phenotype. Introduction of PA3719 into wild‐type P. aeruginosa on a multicopy plasmid was, in fact, sufficient to promote elevated MexAB‐OprM expression and multidrug resistance characteristic of a nalC strain. Thus, the nalC (PA3721) mutation serves only to enhance PA3720‐PA3719 expression, with expression of PA3719 (encodes a 53 amino acid protein of predicted pI 10.4) directly or indirectly impacting MexAB‐OprM expression. Intriguingly, nalC strains produce markedly elevated levels of stable MexR protein suggesting that PA3720‐PA3719 hyperexpression somehow modulates MexR repressor activity. The deduced products of PA3720‐PA3719 show no homology to sequences presently in the GenBank databases, however, and as such provide no clues as to how this might occur.
Repression of the RcsC‐YojN‐RcsB phosphorelay by the IgaA protein is a requisite for Salmonella virulence
Authors: Domínguez‐Bernal Gustavo , Pucciarelli M. Graciela , Ramos‐Morales Francisco , García‐Quintanilla Meritxell , Cano David A. , Casadesús Josep , García‐del Portillo Francisco
Published: August 2004

Bacterial pathogenesis relies on regulators that activate virulence genes. Some of them act, in addition, as repressors of specific genes. Intracellular‐growth‐attenuator‐A (IgaA) is a Salmonella enterica membrane protein that prevents overactivation of the RcsC‐YojN‐RcsB regulatory system. This negative control is critical for growth because disruption of the igaA gene is only possible in rcsC, yojN or rcsB strains. In this work, we examined the contribution of this regulatory circuit to virulence. Viable igaA point mutant alleles were isolated and characterized. These alleles encode IgaA variants leading to different levels of activation of the RcsC‐YojN‐RcsB system. IgaA‐mediated repression of the RcsB‐YojN‐RcsC system occurred at the post‐translational level, as shown by chromosomal epitope tagging of the rcsC, yojN and rcsB genes. The activity of the RcsC‐YojN‐RcsB system, monitored with the product of a tagged gmd‐3xFLAG gene (positively regulated by RcsC‐YojN‐RcsB), was totally abolished by wild‐type bacteria in mouse target organs. Such tight repression occurred only in vivo and was mediated by IgaA. Shutdown of the RcsC‐YojN‐RcsB system is a requisite for Salmonella virulence since all igaA point mutant strains were highly attenuated. The degree of attenuation correlated to that of the activation status of RcsC‐YojN‐RcsB. In some cases, the attenuation recorded was unprecedented, with competitive index (CI) values as low as 10−6. Strikingly, IgaA is a protein absolutely dispensable for virulence in mutant strains having a non‐functional RcsC‐YojN‐RcsB system. To our knowledge, IgaA exemplifies the first protein that contributes to virulence by exclusively acting as a negative regulator upon host colonization.
Regulatory networks affected by iron availability in Candida albicans
Authors: Lan Chung‐Yu , Rodarte Gabriel , Murillo Luis A. , Jones Ted , Davis Ronald W. , Dungan Jan , Newport George , Agabian Nina
Published: August 2004

Iron, an essential element for almost every organism, serves as a regulatory signal for the expression of virulence determinants in many prokaryotic and eukaryotic pathogens. Using a custom Affymetrix GeneChip© representing the entire Candida albicans genome, we examined the changes in genome‐wide gene expression in this opportunistic pathogen as a function of alterations in environmental concentrations of iron. A total of 526 open reading frame (ORF) transcripts are more highly expressed when the levels of available iron are low, while 626 ORF transcripts are more highly expressed in high‐iron conditions. The transcripts dominantly affected by iron concentration range from those associated with cell‐surface properties to others which affect mitochondrial function, iron transport and virulence‐related secreted hydrolases. Moreover gene expression as assayed in DNA microarrays confirms and extends reports of alterations in cell‐surface antigens and drug sensitivity correlated with iron availability. To understand how these genes and pathways might be regulated, we isolated a gene designated SFU1 that encodes a homologue of the Ustilago maydis URBS1, a transcriptional repressor of siderophore uptake/biosynthesis. Comparisons between wild‐type and SFU1‐null mutant strains revealed 139 potential target genes of Sfu1p; many of which are iron‐responsive. Together, these results not only expand our understanding of global iron regulation in C. albicans, but also provide insights into the potential role of iron availability in C. albicans virulence.
The sirodesmin biosynthetic gene cluster of the plant pathogenic fungus Leptosphaeria maculans
Authors: Gardiner Donald M. , Cozijnsen Anton J. , Wilson Leanne M. , Pedras M. Soledade C. , Howlett Barbara J.
Published: August 2004

Sirodesmin PL is a phytotoxin produced by the fungus Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). This phytotoxin belongs to the epipolythiodioxopiperazine (ETP) class of toxins produced by fungi including mammalian and plant pathogens. We report the cloning of a cluster of genes with predicted roles in the biosynthesis of sirodesmin PL and show via gene disruption that one of these genes (encoding a two‐module non‐ribosomal peptide synthetase) is essential for sirodesmin PL biosynthesis. Of the nine genes in the cluster tested, all are co‐regulated with the production of sirodesmin PL in culture. A similar cluster is present in the genome of the opportunistic human pathogen Aspergillus fumigatus and is most likely responsible for the production of gliotoxin, which is also an ETP. Homologues of the genes in the cluster were also identified in expressed sequence tags of the ETP producing fungus Chaetomium globosum. Two other fungi with publicly available genome sequences, Magnaporthe grisea and Fusarium graminearum, had similar gene clusters. A comparative analysis of all four clusters is presented. This is the first report of the genes responsible for the biosynthesis of an ETP.
Dimerization properties of TraM, the antiactivator that modulates TraR‐mediated quorum‐dependent expression of the Ti plasmid tra genes
Authors: Qin Yinping , Smyth Audra J. , Su Shengchang , Farrand Stephen K.
Published: August 2004

TraM, an 11.2 kDa antiactivator, modulates the acyl‐homoserine lactone‐mediated autoinduction of Ti plasmid conjugative transfer by interacting directly with TraR, the quorum‐sensing transcriptional activator. Most antiactivators and antisigma factors examined to date act in dimer form. However, whether, and if so, how TraM dimerizes is unknown. Analyses based on a genetic assay using fusions of TraM to the λ cI DNA binding domain, and biochemical assays using chemical crosslinking and gel filtration chromatography showed that TraM forms homodimers. Although SDS‐PAGE studies suggested that the lone cysteine residue at position 71 was involved in interprotomer disulfide‐bridging in TraM, altering Cys‐71 to a serine did not significantly affect dimerization or the antiactivator activity of this mutant protein when expressed at wild‐type levels in vivo. Analysis of N‐terminal, C‐terminal, and internal deletion mutants of TraM identified two regions of the protein involved in dimerization; one located within a segment between residues 20 and 50, and the other located to a segment between residues 67 and 96. Both regions are required for formation of fully stable dimers. Analysis of the activity of these deletion mutants in vivo, and their ability to bind TraR and to disrupt TraR‐DNA complexes in vitro, suggests that while the internal segment of the protein is required for dimerization, determinants located at the far C‐terminus and beginning at between residues 10 and 20 at the N‐terminus play a role in TraR binding and antiactivator function. When co‐expressed with λ cI′::TraR fusions, wild‐type TraM mediated quormone‐independent dimerization of the transcriptional activator, suggesting that dimers of TraM can multimerize TraR.
A novel ERK‐like, CRK‐like protein kinase that modulates growth in Trypanosoma brucei via an autoregulatory C‐terminal extension
Authors: Ellis James , Sarkar Mitali , Hendriks Edward , Matthews Keith
Published: August 2004

The protozoan parasite Trypanosoma brucei undergoes a complex developmental cycle coordinated with cell cycle control. These processes in eukaryotes are frequently regulated through mitogen‐activated protein kinases (MAPKs) and cyclin‐dependent protein kinases (CDKs), respectively. We have discovered a novel protein kinase which shares features of both ERK‐type MAPKs and CDKs (T. brucei ERK‐like, CDK‐like protein kinase). This molecule, named TbECK1, is similar to the unusual mammalian KKIAMRE protein kinase family. Moreover, TbECK1 possesses a long C‐terminal extension reminiscent of those found in mammalian ERK5, ERK7 and ERK8. Expression analyses demonstrate that TbECK1 is constitutively expressed during the trypanosome life cycle at both RNA and protein level. In transgenic parasites we demonstrate that expression of a mutant of TbECK1 that lacks the C‐terminal extension produces a slow growth phenotype, associated with the appearance of cells with aberrant karyotypes. Using this as an assay we further demonstrate that the phenotype is dependent upon the potential for catalytic activity of TbECK1 and on the integrity of at least one of the phosphorylable amino acids in its phosphorylation lip. C‐terminal extensions are a common feature of kinetoplastid protein kinases. Our results demonstrate for the first time that this domain has a regulatory function.
Tyrosine site‐specific recombinases mediate DNA inversions affecting the expression of outer surface proteins of Bacteroides fragilis
Authors: Weinacht Katja G. , Roche Hazeline , Krinos Corinna M. , Coyne Michael J. , Parkhill Julian , Comstock Laurie E.
Published: August 2004

The chromosome of Bacteroides fragilis has been shown to undergo 13 distinct DNA inversions affecting the expression of capsular polysaccharides and mediated by a serine site‐specific recombinase designated Mpi. In this study, we demonstrate that members of the tyrosine site‐specific recombinase family, conserved in B. fragilis, mediate additional DNA inversions of the B. fragilis genome. These DNA invertases flip promoter regions in their immediate downstream region. The genetic organization of the genes regulated by these invertible promoter regions suggests that they are operons and many of the products are predicted to be outer membrane proteins. Phenotypic analysis of a deletion mutant of one of these DNA invertases, tsr15 (aapI), which resulted in the promoter region for the downstream genes being locked ON, confirmed the synthesis of multiple surface proteins by this operon. In addition, this deletion mutant demonstrated an autoaggregative phenotype and showed significantly greater adherence than wild‐type organisms in a biofilm assay, suggesting a possible functional role for these phase‐variable outer surface proteins. This study demonstrates that DNA inversion is a universal mechanism used by this commensal microorganism to phase vary expression of its surface molecules and involves at least three conserved DNA invertases from two evolutionarily distinct families.
Analysis of the Frz signal transduction system of Myxococcus xanthus shows the importance of the conserved C‐terminal region of the cytoplasmic chemoreceptor FrzCD in sensing signals
Authors: Bustamante Víctor H. , Martínez‐Flores Irma , Vlamakis Hera C. , Zusman David R.
Published: August 2004

The Frz chemosensory system controls directed motility in Myxococcus xanthus by regulating cellular reversal frequency. M. xanthus requires the Frz system for vegetative swarming on rich media and for cellular aggregation during fruiting body formation on starvation media. The Frz signal transduction pathway is formed by proteins that share homology with chemotaxis proteins from enteric bacteria, which are encoded in the frzA‐F putative operon and the divergently transcribed frzZ gene. FrzCD, the Frz system chemoreceptor, contains a conserved C‐terminal module present in methyl‐accepting chemotaxis proteins (MCPs); but, in contrast to most MCPs, FrzCD is localized in the cytoplasm and the N‐terminal region of FrzCD does not contain transmembrane or sensing domains, or even a linker region. Previous work on the Frz system was limited by the unavailability of deletion strains. To understand better how the Frz system functions, we generated a series of in‐frame deletions in each of the frz genes as well as regions encoding the N‐terminal portion of FrzCD. Analysis of mutants containing these deletions showed that FrzCD (MCP), FrzA (CheW) and FrzE (CheA–CheY) control vegetative swarming, responses to repellents and directed movement during development, thus constituting the core components of the Frz pathway. FrzB (CheW), FrzF (CheR), FrzG (CheB) and FrzZ (CheY–CheY) are required for some but not all responses. Furthermore, deletion of ≈ 25 amino acids from either end of the conserved C‐terminal region of FrzCD results in a constitutive signalling state of FrzCD, which induces hyper‐reversals with no net cell movement. Surprisingly, deletion of the N‐terminal region of FrzCD shows only minor defects in swarming. Thus, signal input to the Frz system must be sensed by the conserved C‐terminal module of FrzCD and not the usual N‐terminal region. These results indicate an alternative mechanism for signal sensing with this cytoplasmic MCP.
Synthesis of group A streptococcal virulence factors is controlled by a regulatory RNA molecule
Authors: Mangold Monika , Siller Maria , Roppenser Bernhard , Vlaminckx Bart J. M. , Penfound Tom A. , Klein Reinhard , Novak Rodger , Novick Richard P. , Charpentier Emmanuelle
Published: August 2004

The capacity of pathogens to cause disease depends strictly on the regulated expression of their virulence factors. In this study, we demonstrate that the untranslated mRNA of the recently described streptococcal pleiotropic effect locus (pel), which incidentally contains sagA, the structural gene for streptolysin S, is an effector of virulence factor expression in group A beta‐haemolytic streptococci (GAS). Our data suggest that the regulation by pel RNA occurs at both transcriptional (e.g. emm, sic, nga) and post‐transcriptional (e.g. SpeB) levels. We could exclude the possibility that the pel phenotype was linked to a polar effect on downstream genes (sagB‐I). Remarkably, the RNA effector is regulated in a growth phase‐dependent fashion and we provide evidence that pel RNA expression is induced by conditioned media.
Measurement of SOS expression in individual Escherichia coli K‐12 cells using fluorescence microscopy
Authors: McCool Jesse D. , Long Edward , Petrosino Joseph F. , Sandler Hilary A. , Rosenberg Susan M. , Sandler Steven J.
Published: August 2004

Many recombination, DNA repair and DNA replication mutants have high basal levels of SOS expression as determined by a sulAp‐lacZ reporter gene system on a population of cells. Two opposing models to explain how the SOS expression is distributed in these cells are: (i) the ‘Uniform Expression Model (UEM)’ where expression is evenly distributed in all cells or (ii) the ‘Two Population Model (TPM)’ where some cells are highly induced while others are not at all. To distinguish between these two models, a method to quantify SOS expression in individual bacterial cells was developed by fusing an SOS promoter (sulAp) to the green fluorescent protein (gfp) reporter gene and inserting it at attλ on the Escherichia coli chromosome. It is shown that the fluorescence in sulAp‐gfp cells is regulated by RecA and LexA. This system was then used to distinguish between the two models for several mutants. The patterns displayed by priA, dnaT, recG, uvrD, dam, ftsK, rnhA, polA and xerC mutants were explained best by the TPM while only lexA (def), lexA3 (ind–) and recA defective mutants were explained best by the UEM. These results are discussed in a context of how the processes of DNA replication and recombination may affect cells in a population differentially.