bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric oxide contributes both to papilla‐based resistance and the hypersensitive response in barley attacked by Blumeria graminis f. sp. hordei
Authors: PRATS ELENA , MUR LUIS A. J. , SANDERSON RUTH , CARVER TIMOTHY L. W.
Published: February 2005

Nonspecific penetration resistance due to papilla formation and race‐specific hypersensitive response (HR) can both contribute to Blumeria graminis resistance in barley. Some effective papillae form even in the susceptible cv. Pallas and the isoline P01 carries the additional Mla1 allele conditioning HR. The NO‐specific stain DAF‐2DA (4,5‐diaminofluorescein‐2‐diacetate) revealed a transient NO generation burst commencing 10 h after inoculation (h.a.i.) in close association with sites of papilla formation in both barley lines. In P01 a burst of NO production throughout some attacked cells was initiated around 10–12 h.a.i. and this preceded whole‐cell autofluorescence indicative of HR. The specificity of DAF‐2DA staining was demonstrated by the suppression of staining following application of the NO scavenger C‐PTIO (1H‐imidazol‐1‐yloxy‐2‐(4‐carboxyphenyl)‐4,5‐dihydro‐4,4,5,5‐tetramethyl‐3‐oxide). In addition, C‐PTIO application increased penetration frequencies in both barley lines, indicating a role for NO in papilla‐based resistance. Furthermore, C‐PTIO application slightly delayed HR in P01 whereas, conversely, application of an NO donor, sodium nitroprusside, slightly accelerated HR in P01 and increased cell death frequency in Pallas. Thus, NO generation is one of the earliest responses of barley epidermal cell defence against B. graminis attack and may be important in both the initiation and the development of effective papillae and cell death due to HR.
Prediction of haemorrhage in the early stage of acute myeloid leukaemia by flow cytometric analysis of platelet function
Authors: Leinoe Eva Birgitte , Hoffmann Marianne Hutchings , Kjaersgaard Erik , Nielsen Joern Dalsgaard , Bergmann Olav Jonas , Klausen Tobias Wirenfeldt , Johnsen Hans Erik
Published: February 2005

Haemorrhage is often responsible for the lethal course of acute myeloid leukaemia (AML). Previously, multiple platelet function defects were identified by flow cytometric analysis of platelet activation markers in AML. The role of flow cytometric analysis of platelet function in characterization of prognostic markers of haemorrhage in AML patients has not been well elucidated. The objective of this prospective study was to analyse platelet function in 50 AML patients at diagnosis and to compare results with clinical bleeding score, graded by common toxicity criteria. Platelet activation markers CD62P, CD42b, CD63 and PAC‐1 were analysed following in vitro activation by thrombin receptor activating peptide. The following plasma haemostasis parameters were measured: soluble P‐selectin, activated partial thromboplastin time, thrombin time, prothrombin time, d‐dimer, fibrinogen, and von Willebrand factor antigen. In a multivariate analysis, P‐selectin (CD62P) <36 molecules of equivalent soluble fluorochrome × 103 (P < 0·0015) and platelet count <40 × 109/l (P = 0·01) were significant predictors of haemorrhage at diagnosis. Haemorrhage at diagnosis predicted grade 3–4 haemorrhage in the first 28 d following diagnosis (P = 0·018). The presented results indicate that low P‐selectin is a prognostic marker of haemorrhage in AML.
The effect of hypoxia and stem cell source on haemoglobin switching
Authors: Daisy Narayan A. , Ersek Adel , Campbell Thomas A. , Colón Donna M. , Pixley John S. , Zanjani Esmail D.
Published: February 2005

This study investigated whether relative changes that accompany the naturally occurring shifts in haematopoietic sites during human development play a role in haemoglobin (Hb) switching or whether Hb switching is innately programmed into cells. CD34+/Lineage− haematopoietic stem/progenitor cells (HSCs) were isolated from human fetal liver (F‐LVR), cord blood (CB), and adult bone marrow (ABM), and the Hb was characterized by flow cytometry on cultures that generated enucleated red cells. All feeder layers (stroma from F‐LVR, ABM, and human fetal aorta) enhanced cell proliferation and erythropoiesis but did not affect Hb type. HSCs from CB and F‐LVR generated the same Hb profile under normoxia and hypoxia. HSCs from ABM had single‐positive HbA and double‐positive HbA and HbF cells at normoxia and almost entirely double‐positive cells at hypoxia. Further characterization of these ABM cultures was determined by following mRNA expression for the transcription factors erythroid Kruppel‐like factor (EKLF) and fetal Kruppel‐like factor (FKLF) as a function of time in cultures under hypoxia and normoxia. The erythroid‐specific isoform of 5‐amino‐levulinate synthase (ALAS2) was also expressed under hypoxic conditions. We conclude that Hb switching is affected by the environment but not all HSCs are preprogrammed to respond.
Childhood paroxysmal nocturnal haemoglobinuria (PNH), a report of 11 cases in the Netherlands
Authors: Van Den Heuvel‐Eibrink M. M. , Bredius R. G. M. , Te Winkel M. L. , Tamminga R. , de Kraker J. , Schouten‐van Meeteren A. Y. N. , Bruin M. , Korthof E. T.
Published: February 2005

Paroxysmal nocturnal haemoglobinuria (PNH) is characterized by intravascular haemolysis, nocturnal haemoglobinuria, thrombotic events, serious infections and bone marrow failure. This acquired disease, caused by a deficiency of glycosylphosphatidylinositol (GPI) anchored proteins on the haematopoietic cells, is rare in children. We describe 11 Dutch paediatric PNH patients (median age: 12 years, range 9–17 years) diagnosed since 1983, seven cases associated with aplastic anaemia (AA), four with myelodysplastic syndrome (MDS). Presenting symptoms were haemorrhagic diathesis (n = 10), palor/tiredness (n = 8), dark urine (n = 1), fever (n = 1) and serious weight loss (n = 1). Treatment consisted of prednisolone (n = 7), anti‐thymocyte globulin (n = 3) and/or androgens (n = 5). Eventually, five patients received a bone marrow transplantation (BMT) (three matched unrelated donors/two matched family donors), of whom four are still alive. PNH, diagnosed by immunophenotypic GPI‐linked anchor protein analysis, should be considered in all children with AA or MDS. BMT should be considered as a therapeutic option in every paediatric PNH patient with BM failure.
GATA1 mutation and trisomy 21 are required only in haematopoietic cells for development of transient myeloproliferative disorder
Authors: Carpenter Emily , Valverde‐Garduno Veronica , Sternberg Alex , Mitchell Chris , Roberts Irene , Vyas Paresh , Vora Ajay
Published: February 2005

Trisomy 21 [Down's syndrome (DS)] and mutations in transcription factor GATA1 predispose neonates to a transient myeloproliferative disorder (TMD) and/or acute megakaryocytic leukaemia (AMKL). The role of trisomy 21 in their pathogenesis is unclear. We previously reported two rare neonates without DS who had TMD, one of whom progressed to AMKL. Trisomy 21 was detected only in blood cells at presentation with TMD/AMKL and disappeared with disease resolution. We now show that the blood cells at presentation of TMD harboured GATA1 genomic DNA mutations, suggesting a requirement for trisomy 21 in haematopoietic cells, rather than other cell types, for development of TMD/AMKL.
Estimating diurnal to annual ecosystem parameters by synthesis of a carbon flux model with eddy covariance net ecosystem exchange observations
Authors: Braswell Bobby H. , Sacks William J. , Linder Ernst , Schimel David S.
Published: February 2005

We performed a synthetic analysis of Harvard Forest net ecosystem exchange of CO2(NEE) time series and a simple ecosystem carbon flux model, the simplified Photosynthesis and Evapo‐Transpiration model (SIPNET). SIPNET runs at a half‐daily time step, and has two vegetation carbon pools, a single aggregated soil carbon pool, and a simple soil moisture sub‐model. We used a stochastic Bayesian parameter estimation technique that provided posterior distributions of the model parameters, conditioned on the observed fluxes and the model equations. In this analysis, we estimated the values of all quantities that govern model behavior, including both rate constants and initial conditions for carbon pools. The purpose of this analysis was not to calibrate the model to make predictions about future fluxes but rather to understand how much information about process controls can be derived directly from the NEE observations. A wavelet decomposition enabled us to assess model performance at multiple time scales from diurnal to decadal. The model parameters are most highly constrained by eddy flux data at daily to seasonal time scales, suggesting that this approach is not useful for calculating annual integrals. However, the ability of the model to fit both the diurnal and seasonal variability patterns in the data simultaneously, using the same parameter set, indicates the effectiveness of this parameter estimation method. Our results quantify the extent to which the eddy covariance data contain information about the ecosystem process parameters represented in the model, and suggest several next steps in model development and observations for improved synthesis of models with flux observations.
Soil microbial responses to experimental warming and clipping in a tallgrass prairie
Authors: Zhang W. , Parker K. M. , Luo Y. , Wan S. , Wallace L. L. , Hu S.
Published: February 2005

Global surface temperature is predicted to increase by 1.4–5.8°C by the end of this century. However, the impacts of this projected warming on soil C balance and the C budget of terrestrial ecosystems are not clear. One major source of uncertainty stems from warming effects on soil microbes, which exert a dominant influence on the net C balance of terrestrial ecosystems by controlling organic matter decomposition and plant nutrient availability. We, therefore, conducted an experiment in a tallgrass prairie ecosystem at the Great Plain Apiaries (near Norman, OK) to study soil microbial responses to temperature elevation of about 2°C through artificial heating in clipped and unclipped field plots. While warming did not induce significant changes in net N mineralization, soil microbial biomass and respiration rate, it tended to reduce extractable inorganic N during the second and third warming years, likely through increasing plant uptake. In addition, microbial substrate utilization patterns and the profiles of microbial phospholipid fatty acids (PLFAs) showed that warming caused a shift in the soil microbial community structure in unclipped subplots, leading to the relative dominance of fungi as evidenced by the increased ratio of fungal to bacterial PLFAs. However, no warming effect on soil microbial community structure was found in clipped subplots where a similar scale of temperature increase occurred. Clipping also significantly reduced soil microbial biomass and respiration rate in both warmed and unwarmed plots. These results indicated that warming‐led enhancement of plant growth rather than the temperature increase itself may primarily regulate soil microbial response. Our observations show that warming may increase the relative contribution of fungi to the soil microbial community, suggesting that shifts in the microbial community structure may constitute a major mechanism underlying warming acclimatization of soil respiration.
Plant and microbial N acquisition under elevated atmospheric CO2 in two mesocosm experiments with annual grasses
Authors: Hu Shuijin , Wu Jiansheng , Burkey Kent O. , Firestone Mary K.
Published: February 2005

The impact of elevated CO2 on terrestrial ecosystem C balance, both in sign or magnitude, is not clear because the resulting alterations in C input, plant nutrient demand and water use efficiency often have contrasting impacts on microbial decomposition processes. One major source of uncertainty stems from the impact of elevated CO2 on N availability to plants and microbes. We examined the effects of atmospheric CO2 enrichment (ambient+370 μmol mol−1) on plant and microbial N acquisition in two different mesocosm experiments, using model plant species of annual grasses of Avena barbata and A. fatua, respectively. The A. barbata experiment was conducted in a N‐poor sandy loam and the A. fatua experiment was on a N‐rich clayey loam. Plant–microbial N partitioning was examined through determining the distribution of a 15N tracer. In the A. barbata experiment, 15N tracer was introduced to a field labeling experiment in the previous year so that 15N predominantly existed in nonextractable soil pools. In the A. fatua experiment, 15N was introduced in a mineral solution [(15NH4)2SO4 solution] during the growing season of A. fatua. Results of both N budget and 15N tracer analyses indicated that elevated CO2 increased plant N acquisition from the soil. In the A. barbata experiment, elevated CO2 increased plant biomass N by ca. 10% but there was no corresponding decrease in soil extractable N, suggesting that plants might have obtained N from the nonextractable organic N pool because of enhanced microbial activity. In the A. fatua experiment, however, the CO2‐led increase in plant biomass N was statistically equal to the reduction in soil extractable N. Although atmospheric CO2 enrichment enhanced microbial biomass C under A. barbata or microbial activity (respiration) under A. fatua, it had no significant effect on microbial biomass N in either experiment. Elevated CO2 increased the colonization of A. fatua roots by arbuscular mycorrhizal fungi, which coincided with the enhancement of plant competitiveness for soluble soil N. Together, these results suggest that elevated CO2 may tighten N cycling through facilitating plant N acquisition. However, it is unknown to what degree results from these short‐term microcosm experiments can be extrapolated to field conditions. Long‐term studies in less‐disturbed soils are needed to determine whether CO2‐enhancement of plant N acquisition can significantly relieve N limitation over plant growth in an elevated CO2 environment.
Linking microbial activity and soil organic matter transformations in forest soils under elevated CO2
Authors: Billings S. A. , Ziegler S. E.
Published: February 2005

Soil organic matter (SOM) dynamics ultimately govern the ability of soil to provide long‐term C sequestration and the nutrients required for ecosystem productivity. Predicting belowground responses to elevated CO2 requires an integrated understanding of SOM transformations and the microbial activity that governs them. It remains unclear how the microorganisms upon which these transformations depend will function in an elevated CO2 world. This study examines SOM transformations and microbial metabolism in soils from the Duke Free Air Carbon Enrichment site in North Carolina, USA. We assessed microbial respiration and net nitrogen (N) mineralization in soils with and without elevated CO2 exposure during a 100‐day incubation. We also traced the depleted C isotopic signature of the supplemental CO2 into SOM and the soils' phospholipid fatty acids (PLFA), which serve as biomarkers for living cells. Cumulative net N mineralization in elevated CO2 soils was 50% that in control soils after a 100‐day incubation. Respiration was not altered with elevated CO2. C : N ratios of bulk SOM did not change with elevated CO2, but incubation data suggest that the C : N ratios of mineralized organic matter increased with elevated CO2. Values of SOM δ13C were depleted with elevated CO2 (−26.7±0.2 vs. −30.2±0.3‰), reflecting the depleted signature of the supplemental CO2. We compared δ13C of individual PLFA with the δ13C of SOM to discern incorporation of the depleted C isotopic signature into soil microbial groups in elevated CO2 plots. PLFA i15:0, a15:0, and 10Met18:0 reflected significant incorporation of recently produced photosynthate, suggesting that the bacterial groups defined by these biomarkers are active metabolizers in elevated CO2 soils. At least one of these groups (actinomycetes, 10Met18:0) specializes in metabolizing less labile substrates. Because control plots did not receive an equivalent 13C tracer, we cannot determine from these data whether this group of organisms was stimulated by elevated CO2 compared with these organisms in control soils. Stimulation of this group, if it occurred in the elevated CO2 plot, would be consistent with a decline in the availability of mineralizable organic matter with elevated CO2, which incubation data suggest may be the case in these soils.
A membrane metalloprotease participates in the sequential degradation of a Caulobacter polarity determinant
Authors: Chen Joseph C. , Viollier Patrick H. , Shapiro Lucy
Published: February 2005

Caulobacter crescentus assembles many of its cellular machines at distinct times and locations during the cell cycle. PodJ provides the spatial cues for the biogenesis of several polar organelles, including the pili, adhesive holdfast and chemotactic apparatus, by recruiting structural and regulatory proteins, such as CpaE and PleC, to a specific cell pole. PodJ is a protein with a single transmembrane domain that exists in two forms, full‐length (PodJL) and truncated (PodJS), each appearing during a specific time period of the cell cycle to control different aspects of polar organelle development. PodJL is synthesized in the early predivisional cell and is later proteolytically converted to PodJS. During the swarmer‐to‐stalked transition, PodJS must be degraded to preserve asymmetry in the next cell cycle. We found that MmpA facilitates the degradation of PodJS. MmpA belongs to the site‐2 protease (S2P) family of membrane‐embedded zinc metalloproteases, which includes SpoIVFB and YluC of Bacillus subtilis and YaeL of Escherichia coli. MmpA appears to cleave within or near the transmembrane segment of PodJS, releasing it into the cytoplasm for complete proteolysis. While PodJS has a specific temporal and spatial address, MmpA is present throughout the cell cycle; furthermore, periplasmic fusion to mRFP1 suggested that MmpA is uniformly distributed around the cell. We also determined that mmpA and yaeL can complement each other in C. crescentus and E. coli, indicating functional conservation. Thus, the sequential degradation of PodJ appears to involve regulated intramembrane proteolysis (Rip) by MmpA.
A PrfA‐regulated bile exclusion system (BilE) is a novel virulence factor in Listeria monocytogenes
Authors: Sleator Roy D. , Wemekamp‐Kamphuis Henrike H. , Gahan Cormac G. M. , Abee Tjakko , Hill Colin
Published: February 2005

The ability to colonize the gall bladder has recently been shown to be an important feature of virulent Listeria monocytogenes (J. Hardy, K. P. Francis, M. DeBoer, P. Chu, K. Gibbs, C. H. Contag. Science 303: 851–853, 2004). We suggest that the cytotoxic effects of bile may be increased upon release from the gall bladder into the upper small intestine, and report the identification of a novel bile exclusion system which plays an essential role in intestinal colonization and virulence of L. monocytogenes. In silico analysis of the L. monocytogenes EGDe genome revealed a two‐gene operon (formerly opuB) exhibiting significant sequence similarity to members of the betaine carnitine choline transporter (BCCT) family. The operon, herein designated bilE (bile Exclusion) is preceded by consensus σA‐ and σB‐dependent promoter‐binding sites and is transcriptionally upregulated at elevated osmolarities and reduced temperatures (stresses known to induce sigB). Furthermore, a significant reduction in the level of bilE transcription was observed in the absence of σB. In addition, we demonstrate an important role for PrfA, the master regulator of virulence potential in L. monocytogenes, in coordinating bilE expression. Computational structural analysis suggests that, rather than functioning as a compatible solute uptake system as was previously believed, BilE is more likely to be an exclusion system, a conclusion substantiated by radiolabelled bile accumulation studies. In addition, functionally inactivating BilE resulted in a five‐log reduction in the ability of the bacterium to tolerate lethal concentrations of bovine bile (oxgall) and also significantly increased sensitivity to physiological concentrations of human bile, a phenotype which translates to a significant reduction in virulence potential when administered to a murine model by the oral route. Thus, this novel bile exclusion locus bilE, coordinately regulated by σB and PrfA, represents a new and important virulence factor in L. monocytogenes.
A large, mobile pathogenicity island confers plant pathogenicity on Streptomyces species
Authors: Kers Johan A. , Cameron Kimberly D. , Joshi Madhumita V. , Bukhalid Raghida A. , Morello Joanne E. , Wach Michael J. , Gibson Donna M. , Loria Rosemary
Published: February 2005

Potato scab is a globally important disease caused by polyphyletic plant pathogenic Streptomyces species. Streptomyces acidiscabies, Streptomyces scabies and Streptomyces turgidiscabies possess a conserved biosynthetic pathway for the nitrated dipeptide phytotoxin thaxtomin. These pathogens also possess the nec1 gene which encodes a necrogenic protein that is an independent virulence factor. In this article we describe a large (325–660 kb) pathogenicity island (PAI) conserved among these three plant pathogenic Streptomyces species. A partial DNA sequence of this PAI revealed the thaxtomin biosynthetic pathway, nec1, a putative tomatinase gene, and many mobile genetic elements. In addition, the PAI from S. turgidiscabies contains a plant fasciation (fas) operon homologous to and colinear with the fas operon in the plant pathogen Rhodococcus fascians. The PAI was mobilized during mating from S. turgidiscabies to the non‐pathogens Streptomyces coelicolor and Streptomyces diastatochromogenes on a 660 kb DNA element and integrated site‐specifically into a putative integral membrane lipid kinase. Acquisition of the PAI conferred a pathogenic phenotype on S. diastatochromogenes but not on S. coelicolor. This PAI is the first to be described in a Gram‐positive plant pathogenic bacterium and is responsible for the emergence of new plant pathogenic Streptomyces species in agricultural systems.
Genetic rescue of a Toxoplasma gondii conditional cell cycle mutant
Authors: White Michael W. , Jerome Maria E. , Vaishnava Shipra , Guerini Michael , Behnke Michael , Striepen Boris
Published: February 2005

Growth rate is a major pathogenesis factor in the parasite Toxoplasma gondii; however, how cell division is controlled in this protozoan is poorly understood. Herein, we show that centrosomal duplication is an indicator of S phase entry while centrosome migration marks mitotic entry. Using the pattern of centrosomal replication, we confirmed that mutant ts11C9 undergoes a bimodal cell cycle arrest that is characterized by two subpopulations containing either single or duplicated centrosomes which correlate with the bipartite genome distribution observed at the non‐permissive temperature. Genetic rescue of ts11C9 was performed using a parental RH strain cDNA library, and the cDNA responsible for conferring temperature resistance (growth at 40°C) was recovered by recombination cloning. A single T. gondii gene encoding the protein homologue of XPMC2 was responsible for genetic rescue of the temperature‐sensitive defect in ts11C9 parasites. This protein is a known suppressor of mitotic defects, and in tachyzoites, TgXPMC2–YFP localized to the parasite nucleus and nucleolus which is consistent with the expected subcellular localization of critical mitotic factors. Altogether, these results demonstrate that ts11C9 is a conditional mitotic mutant containing a single defect which influences two distinct control points in the T. gondii tachyzoite cell cycle.
The Aspergillus pH‐responsive transcription factor PacC regulates virulence
Authors: Bignell Elaine , Negrete‐Urtasun Susana , Calcagno Ana Maria , Haynes Ken , Arst Herbert N. , Rogers Tom
Published: February 2005

The ability of a pathogen to adapt to the host environment is usually required for the initiation of disease. Here we have investigated the importance of the Aspergillus nidulans PacC‐mediated pH response in the pathogenesis of pulmonary aspergillosis. Using mutational analysis, we demonstrate that, in neutropenic mice, elimination of the A. nidulans pH‐responsive transcription factor PacC, blocking the ambient pH signal transduction pathway or prevention of PacC proteolytic processing acutely attenuates virulence. Infections caused by these alkali‐sensitive mutants are characterized by limited growth in vivo and a reduction of inflammatory cell infiltration. In stark contrast, constitutive activation of PacC causes increased mortality marked by extensive fungal invasive growth. PacC action is therefore required for, and able to enhance virulence, demonstrating that the A. nidulans pH‐responsive transcription factor PacC plays a pivotal role in pulmonary pathogenesis.
Supportive and inhibitory elements of a putative PrfA‐dependent promoter inListeria monocytogenes
Authors: Luo Qin , Herler Michael , Müller‐Altrock Stefanie , Goebel Werner
Published: February 2005

Elements essential for PrfA‐dependent transcription were analysed on two promoters ofListeria monocytogenes, the PrfA‐dependent promoter of the phospholipase geneplcA(PplcA) and a putative promoter of thearoAgene (ParoA2) which contains a similar PrfA‐binding site and a similar −10 box as PplcAbut does not function as PrfA‐dependent promoter. We constructed a series of hybridplcA‐aroApromoters by exchanging corresponding sequence elements of these two ‘promoters’. The results showed that the two critical elements of PrfA‐dependent promoters, the PrfA‐box and the −10 box, can be functionally exchanged as long as the distance in between is maintained to 22 or 23 bp. However, the interspace sequence and the sequence downstream of the −10 box of ParoA2were strongly inhibitory for PrfA‐dependent transcription. A detailed analysis of these two sequences revealed that the RNA polymerase binding site being part of the actualin vivoandin vitrousedaroApromoter (ParoA1) and a sequence immediately downstream of the putative −10 site, possibly blocking the formation of the open complex, were responsible for the inhibition of PrfA‐dependent transcription from ParoA2. Taking into consideration the lessons learned from this study we were able to construct a functional PrfA‐dependentaroApromoter.
Defence of Rhizobium etli bacteroids against oxidative stress involves a complexly regulated atypical 2‐Cys peroxiredoxin
Authors: Dombrecht Bruno , Heusdens Christophe , Beullens Serge , Verreth Christel , Mulkers Esther , Proost Paul , Vanderleyden Jos , Michiels Jan
Published: February 2005

In general, oxidative stress, the consequence of an aerobic lifestyle, induces bacterial antioxidant defence enzymes. Here we report on a peroxiredoxin of Rhizobium etli, prxS, strongly expressed under microaerobic conditions and during the symbiotic interaction with Phaseolus vulgaris. The microaerobic induction of the prxS‐rpoN2 operon is mediated by the alternative sigma factor RpoN and the enhancer‐binding protein NifA. The RpoN‐dependent promoter is also active under low‐nitrogen conditions through the enhancer‐binding protein NtrC. An additional symbiosis‐specific weak promoter is located between prxS and rpoN2. Constitutive expression of prxS confers enhanced survival and growth to R. etli in the presence of H2O2. Single prxS mutants are not affected in their symbiotic abilities or defence response against oxidative stress under free‐living conditions. In contrast, a prxS katG double mutant has a significantly reduced (>40%) nitrogen fixation capacity, suggesting a functional redundancy between PrxS and KatG, a bifunctional catalase‐peroxidase. In vitro assays demonstrate  the  reduction  of  PrxS  protein  by  DTT and thioredoxin. PrxS displays substrate specificity towards H2O2 (Km = 62 µM) over alkyl hydroperoxides (Km > 1 mM). Peroxidase activity is abolished in both the peroxidatic (C56) and resolving (C156) cysteine PrxS mutants, while the conserved C81 residue is required for proper folding of the protein. Resolving of the R. etli PrxS peroxidatic cysteine is probably an intramolecular process and intra‐ and intersubunit associations were observed. Taken together, our data support, for the first time, a role for an atypical 2‐Cys peroxiredoxin against oxidative stress in R. etli bacteroids.
Global analysis of the core cell cycle regulators of Arabidopsis identifies novel genes, reveals multiple and highly specific profiles of expression and provides a coherent model for plant cell cycle control
Authors: Menges Margit , De Jager Sarah M. , Gruissem Wilhelm , Murray James A.H.
Published: February 2005

Arabidopsis has over 80 genes encoding conserved and plant‐specific core cell cycle regulators, but in most cases neither their timing of expression in the cell cycle is known nor whether they represent redundant and/or tissue‐specific functions. Here we identify novel cell cycle regulators, including new cyclin‐dependent kinases related to the mammalian galactosyltransferase‐associated protein kinase p58, and new classes of cyclin‐like and CDK‐like proteins showing strong tissue specificity of expression. We analyse expression of all cell cycle regulators in synchronized Arabidopsis cell cultures using multiple approaches including Affymetrix microarrays, massively parallel signature sequencing and real‐time reverse transcriptase polymerase chain reaction, and in plant material using the results of over 320 microarray experiments. These global analyses reveal that most core cell cycle regulators are expressed across almost all tissues and more than 85% are expressed at detectable levels in the cell suspension culture, allowing us to present a unified model of transcriptional regulation of the plant cell cycle. Characteristic patterns of D‐cyclin expression in early and late G1 phase, either limited to the re‐entry cycle or continuously oscillating, suggest that several CYCD genes with strong oscillatory regulation in late G1 may play the role of cyclin E in plants. Alone amongst the six groups of A and B type cyclins, members of CYCA3 peak in S‐phase suggest it is a major component of S‐phase kinases, whereas others show a peak in G2/M. 82 genes share this G2/M regulatory pattern, about half being new candidate mitotic genes of previously unknown function.
A novel isoform of glucan, water dikinase phosphorylates pre‐phosphorylated α‐glucans and is involved in starch degradation in Arabidopsis
Authors: Baunsgaard Lone , Lütken Henrik , Mikkelsen René , Glaring Mikkel A. , Pham Tam T. , Blennow Andreas
Published: February 2005

An Arabidopsis thaliana gene encoding a homologue of the potato α‐glucan, water dikinase GWD, previously known as R1, was identified by screening the Arabidopsis genome and named AtGWD3. The AtGWD3 cDNA was isolated, heterologously expressed and the protein was purified to apparent homogeneity to determine the enzymatic function. In contrast to the potato GWD protein, the AtGWD3 primarily catalysed phosphorylation at the C‐3 position of the glucose unit of preferably pre‐phosphorylated amylopectin substrate with long side chains. An Arabidopsis mutant, termed Atgwd3, with downregulated expression of the AtGWD3 gene was analysed. In Atgwd3 the amount of leaf starch was constantly higher than wild type during the diurnal cycle. Compared with wild‐type leaf starch, the level of C‐3 phosphorylation of the glucosyl moiety of starch in this mutant was reduced. Taken together, these data indicate that the C‐3 linked phospho‐ester in starch plays a so far unnoticed specific role in the degradation of transitory starch.
ROS perception in Arabidopsis thaliana: the ozone‐induced calcium response
Authors: Evans Nicola H. , McAinsh Martin R. , Hetherington Alistair M. , Knight Marc R.
Published: February 2005

Ozone is responsible for more crop losses than any other air pollutant. The changes in gene expression, which occur in plants in response to ozone, have been well characterized, yet little is known about how ozone is perceived or the signal transduction steps that follow. The earliest characterized response to ozone is an elevation in cytosolic‐free calcium, which takes place within seconds of exposure. In this study, the calcium response to ozone was investigated in Arabidopsis thaliana seedlings using a variety of fumigation protocols. Ozone elicited distinct calcium responses in the aerial tissue and roots of seedlings. The calcium response in the cotyledons and leaves was biphasic and sensitive to the rate at which the ozone concentration increased. The response in the root was monophasic and insensitive to the rate of increase in ozone concentration. Experiments utilizing inhibitors of antioxidant metabolism demonstrated that the magnitude of the first peak in calcium in the aerial tissues was dependent upon the redox status of the plant. Seedlings were shown to be able to distinguish between ozone and hydrogen peroxide, producing a calcium signal in response to one of these reactive oxygen species (ROS) when they had become refractory to the other. Pre‐treatment with ozone altered the calcium response to hydrogen peroxide and vice versa, indicating that the calcium response to a given ROS may reflect the stress history of the plant. These data suggest ROS signalling is more sophisticated than previously realized and raise questions over current models of ozone perception.
Cyclic enkephalin analogs containing various para‐substituted phenylalanine derivatives in place of Tyr1 are potent opioid agonists*
Authors: Weltrowska G. , Lemieux C. , Chung N.N. , Schiller P.W.
Published: February 2005

Abstract:  The cyclic enkephalin analog H‐Tyr‐c[d‐Cys‐Gly‐Phe(pNO2)‐d‐Cys]NH2 is a highly potent opioid agonist with IC50s of 35 pm and 19 pm in the guinea‐pig ileum (GPI) and mouse vas deferens (MVD) assays, respectively. The Phe1‐analog of this peptide showed 370‐fold and 6790‐fold lower agonist potency in the GPI and MVD assays, respectively, indicating the importance of the Tyr1 hydroxyl‐group in the interaction with μ and δ opioid receptors. In the present study, the effect of various substituents (‐NH2, ‐NO2, ‐CN, ‐CH3, ‐COOH, ‐COCH3, ‐CONH2) introduced in the para‐position of the Phe1‐residue of H‐Phe‐c[d‐Cys‐Gly‐Phe(pNO2)‐d‐Cys]NH2 on the in vitro opioid activity profile was examined. Most analogs showed enhanced μ and δ agonist potencies in the two bioassays, except for the Phe(pCOOH)1‐analog, which was weakly active, probably as a consequence of the negative charge. The most potent compounds were the Phe(pCOH3)1‐ and the Phe(pCONH2)1‐analogs. The latter compound showed subnanomolar μ and δ agonist potencies and represents the most potent enkephalin analog lacking the Tyr1 hydroxyl‐group reported to date. Taken together, these results indicate that various substituents introduced in the para‐position of Phe1 enhance opioid activity via hydrogen bonding or hydrophobic interactions with the receptor. Comparison with existing structure‐activity relationship on phenolic hydroxyl replacements in morphinans indicates that these nonpeptide opiates and some of the cyclic enkephalin analogs described here may have different modes of binding to the receptor.
On the orange color of Z‐Trp‐ONPo*
Authors: Moretto A. , Formaggio F. , Crisma M. , Toniolo C. , Kaptein B. , Broxterman Q.B.
Published: February 2005

Abstract:  Out of all nitrophenyl esters of Nα‐protected α‐amino acids Z‐Trp‐ONPo is the only one which is deep orange colored in the crystalline state. Any change in Nα‐protection, nature of amino acid, spatial separation between Trp and the ester‐group or position of the nitro‐substitutent in the aromatic ring of the ester function results in a loss of this characteristic property. We solved the molecular and crystal structure of Z‐l‐Trp‐ONPo by X‐ray diffraction analysis and investigated its color changes and visible (vis) and infrared (IR) absorption spectra in the solid state as a function of the amino acid derivative/KBr (w/w) ratio in the pellets. This investigation was extended to toluene solutions of different Z‐Trp‐ONPo concentrations by use of vis absorption and proton magnetic resonance spectroscopic techniques. The onset of the orange color correlates closely with the appearance of a concentration‐dependent absorption band near 500 nm and concentration‐dependent shifts of the urethane and indole NH proton resonances. Our observations can be explained by the formation of an intermolecular charge transfer complex involving the Trp indole and the ‐ONPo nitrophenyl as the donor and the acceptor moieties, respectively.
Syntheses of optically pure, conformationally constrained, and highly hydrophobic unusual amino acids: 2‐amino‐3, 3‐diarylpropionic acids*
Authors: Lin J. , Liao S. , Hruby V.J.
Published: February 2005

Abstract:  A series of optically pure, conformationally constrained, and highly hydrophobic unusual aromatic amino acids, 2‐amino‐3,3‐diarylpropionic acids, were synthesized via asymmetric 1,4‐Michael addition reaction/azidation reactions in seven steps with overall yields of 20–30%.
Synthesis of dermorphin‐(1–4) derivatives catalyzed by proteases in organic solvents*
Authors: Shen H.‐Y. , Tian G.‐L. , Ye Y.‐H.
Published: February 2005

Abstract:  In order to extend the use of proteases to organic synthesis and seek the rules of enzymatic reactions in organic media, we focused on unnatural substrates for proteases to form amide bonds. In this paper, the study of unnatural substrates containing d‐amino acid residue, which act as acyl acceptors as well as acyl donors for proteases in organic media, is reported. Dermorphin is a heptapeptide (H‐Tyr‐d‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH2) with potent analgesic activity. The N‐terminal tetrapeptide is the minimum sequence that retains dermorphin activity, and is selected as the model compound in our study. Two dermorphin‐(1–4) derivatives, Boc‐Tyr‐d‐Ala‐Phe‐Gly‐N2H2Ph and Boc‐Tyr‐d‐Ala‐Phe‐Gly‐NH2, which contained a d‐amino acid residue, were synthesized by proteases in organic media for the first time. The synthesis of these two dermorphin‐(1–4) derivatives could be catalyzed by subtilisin with Boc‐Tyr‐d‐Ala‐OCH2CF3 as an acyl donor substrate in AcOEt. The synthesis of dermorphin‐(1–2) derivative Boc‐Tyr‐d‐Ala‐N2H2Ph was catalyzed by α‐chymotrypsin in different organic solvents and d‐Ala‐N2H2Ph was used as an acyl acceptor substrate. Factors influencing the above enzymatic reactions were systematically studied.
Facile degradative lactonization of Gln‐Arg and Gln‐Phe hydroxyethylene dipeptide derivatives*
Authors: Haug B.E. , Brewer M. , Rich D.H.
Published: February 2005

Abstract:  We have found that hydroxyethylene (HE) dipeptide analogs of Gln‐Arg and Gln‐Phe are unusually susceptible to acid catalyzed lactonization. The synthesis of substrate‐based transition state analog inhibitors of botulinum neurotoxin metalloprotease inhibitors that contain the Gln‐Arg or the Gln‐Phe HE units is complicated by this facile degradative lactonization.
Applications of topologically segregated bilayer beads in ‘one‐bead one‐compound’ combinatorial libraries
Authors: Wang X. , Peng L. , Liu R. , Xu B. , Lam K.S.
Published: February 2005

Abstract:  We recently reported the use of a biphasic approach to generate topologically segregated bilayer beads. In generating ‘one‐bead one‐compound’ (OBOC) combinatorial libraries, novel encoding methods have been applied to these beads such as the testing library compound and the coding tags residing on the outer layer and inner core of each bead, respectively. In this report, we further exploit these bilayer beads by preparing target bead‐libraries with low concentration of random peptides on the outer layer, and full substitution of coding peptides in the bead interior. The low concentration of peptide on the bead surface enables us to greatly increase the stringency of screening so that higher affinity ligands can easily be identified. Full substitution of the inner core of the beads enables us to have enough coding peptides inside the bead for direct microsequencing with Edman chemistry. The biphasic approach of preparing bilayer beads can be carried out at any point during the library construction. Therefore, the nonsequencable or fixed structures of the peptides can be bypassed in the coding tags. As a result, peptide libraries that otherwise cannot be sequenced can now be sequenced, and peptide segments with fixed residues within the libraries can be bypassed so that the microsequencing time can be significantly shortened. Furthermore, peptides with a branch of random sequence in the middle of a fixed peptide chain can be encoded with just the random sequence in the bead interior. We have successfully applied these novel OBOC library concepts in the optimization of cell‐surface ligands for a human T‐cell leukemia, Jurkat, cell line.
Bioactive N‐terminal undecapeptides derived from parathyroid hormone: the role of α‐helicity*
Authors: Barazza A. , Wittelsberger A. , Fiori N. , Schievano E. , Mammi S. , Toniolo C. , Alexander J.M. , Rosenblatt M. , Peggion E. , Chorev M.
Published: February 2005

Abstract:  The N‐terminal 1–34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses in bone characteristic of the native intact PTH. Recent studies have demonstrated that N‐terminal fragments presenting the principal activating domain such as PTH(1–11) and PTH(1–14) with helicity‐enhancing substitutions yield potent analogues with PTH(1–34)‐like activity. To further investigate the role of α‐helicity on biological potency, we designed and synthesized by solid‐phase methodology the following hPTH(1–11) analogues substituted at positions 1 and/or 3 by the sterically hindered and helix‐promoting Cα‐tetrasubstituted α‐amino acids α‐amino isobutyric acid (Aib), 1‐aminocyclopentane‐1‐carboxylic acid (Ac5c) and 1‐aminocyclohexane‐1‐carboxylic acid (Ac6c): Ac5c‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH2 (I); Aib‐V‐Ac5c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH2 (II); Ac6c‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH2 (III); Aib‐V‐Ac6c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH2 (IV); Aib‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH2 (V); S‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH2 (VI), S‐V‐Ac5c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH2 (VII); Ac5c‐V‐S‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH2 (VIII); Ac6c‐V‐S‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH2 (IX); Ac5c‐V‐Ac5c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH2 (X); Ac6c‐V‐Ac6c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH2 (XI). All analogues were biologically evaluated and conformationally characterized in 2,2,2‐trifluoroethanol (TFE) solution by circular dichroism (CD). Analogues I–V, which cover the full range of biological activity observed in the present study, were further conformationally characterized in detail by nuclear magnetic resonance (NMR) and computer simulations studies. The results of ligand‐stimulated cAMP accumulation experiments indicated that analogues I and II are active, analogues III, VI and VII are very weakly active and analogues IV, V, VIII–XI are inactive. The most potent analogue, I exhibits biological activity 3500‐fold higher than that of the native PTH(1–11) and only 15‐fold weaker than that of the native sequence hPTH(1–34). Remarkably, the two most potent analogues, I and II, and the very weakly active analogues, VI and VII, exhibit similar helix contents. These results indicate that the presence of a stable N‐terminal helical sequence is an important but not sufficient condition for biological activity.
Residential Property Values and Community Right‐to‐Know Laws: Has the Toxics Release Inventory Had an Impact?
Authors: DECKER CHRISTOPHER S. , NIELSEN DONALD A. , SINDT ROGER P.
Published: February 2005

ABSTRACT  This paper uses a cross‐sectional hedonic pricing model to investigate the relationship between the U.S. Environmental Protection Agency's (EPA) Toxics Release Inventory (TRI) data releases and the prices of single‐family residences within postal zip code areas situated in Omaha, Nebraska's Douglas County. The model employs demographic data from the 2000 Census, toxic chemical release data for the year 2000 from the EPA's TRI database, as well as other pollution variables for each of the zip code areas comprising Douglas County. The model also employs house‐specific data supplied by the Douglas County Assessor's Office on dwelling characteristics for homes sold in Douglas County in the year 2000. The findings indicate that, when controlling for relevant socioeconomic variables, TRI pollutant releases are significant determinants of residential housing values. The results should be of interest to anyone in the real estate industry, including tax assessors, appraisers, mortgage lenders, and property owners.
Low atmospheric oxygen avoids maturation, senescence and cell death of murine mesencephalic neural precursors
Authors: Milosevic Javorina , Schwarz Sigrid C. , Krohn Knut , Poppe Monika , Storch Alexander , Schwarz Johannes
Published: February 2005

The efficient generation of specific brain cells in vitro may serve as a source of cells for brain repair in several devastating neurological diseases. Production of dopaminergic neurons from precursor cells for transplantation in Parkinson's disease has become a major research goal. We found that murine mesencephalic neurospheres were viable and proliferated, preserved telomerase activity, pluripotency and dopaminergic commitment for many weeks when cultured in 3% O2, whereas exposing these cells to 21% oxygen prohibited long‐term expansion. Microarray data suggest that a variety of genes related to the cell cycle, cell maturation and apoptosis are differentially regulated in midbrain‐derived precursors cultured in 3 versus 21% oxygen after 1–2 months. Taken together, we hypothesize that sustained high oxygen has deleterious effects on the self‐renewal capacity of mesencephalic neural precursors, possibly accelerating maturation and senescence resulting in overall cell loss. Gene regulation governed by low oxygen tension may be relevant to the normal development and survival of midbrain neurons.
Effect of apocalmodulin on recombinant human brain glutamic acid decarboxylase
Authors: Jin Hong , Sha Di , Wei Jianning , Davis Kathleen M. , Wu Heng , Jin Ying , Wu Jang‐Yen
Published: February 2005

In this work, we report that the recombinant glutathione S‐transferase (GST)‐human l‐glutamic acid decarboxylase (HGAD) isoforms, 65‐kDa l‐glutamic acid decarboxylase (GAD) (GST‐HGAD65) fusion protein or free truncated HGAD65, were activated by apocalmodulin (ApoCaM) to an extent of 60%. Both truncated forms of GAD67 (tGAD67), HGAD67(Δ1–70) and HGAD67(Δ1–90), were markedly activated by ApoCaM to an extent of 141 and 85%, respectively, while GST‐HGAD67 was not significantly affected. The activation appears to be due to an increase of GAD affinity for its cofactor, pyridoxal phosphate (PLP). This conclusion is based on the following observations. Firstly, the Vmax of GAD was increased when ApoCaM was present whereas the affinity for the substrate, glutamate, was not affected. Secondly, the affinity of GAD for PLP was increased in the presence of ApoCaM. Thirdly, results from calmodulin‐agarose affinity column chromatography studies indicated a direct interaction or binding between ApoCaM and GAD. Fourthly, ApoCaM was found to be copurified with GAD65/GAD67 by anti‐GAD65/67 immunoaffinity column using rat brain extract. Hence, it is proposed that a conformational change is induced when ApoCaM interacts with GAD65 or tGAD67, resulting in an increase of GAD affinity for PLP and the activation of GAD. The physiological significance of the interaction between GAD and ApoCaM is discussed.