bluuebunny/crossref_metadata_2025 huggingface dataset:
The DNA damage signaling pathway is a critical mediator of oncogene-induced senescence
Authors: Mallette Frédérick A. , Gaumont-Leclerc Marie-France , Ferbeyre Gerardo
Published: January 2007

Here we report that RNA interference against ATM inhibited p53 accumulation in cells expressing oncogenic STAT5 and cooperated with Rb inactivation to suppress STAT5A-induced senescence. Knocking down ATM was also effective to bypass E2F1-induced senescence and in combination with Rb inactivation, inhibited RasV12-induced senescence. Cells that senesced in response to ca-STAT5A or RasV12 accumulated DNA damage foci and activated ATM, ATR, Chk1, and Chk2, indicating that aberrant oncogene activation induces a DNA damage signaling response. Intriguingly, bypassing oncogene-induced senescence by inactivation of p53 and Rb did not eliminate the accumulation of oncogene-induced DNA damage foci (ODDI), suggesting a mechanism that may limit transformation in immortalized cells.
pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16INK4a tumor suppressor gene
Authors: Kotake Yojiro , Cao Ru , Viatour Patrick , Sage Julien , Zhang Yi , Xiong Yue
Published: January 2007

Genetic studies have demonstrated that Bmi1 promotes cell proliferation and stem cell self-renewal with a correlative decrease of p16INK4a expression. Here, we demonstrate that Polycomb genes EZH2 and BMI1 repress p16 expression in human and mouse primary cells, but not in cells deficient for pRB protein function. The p16 locus is H3K27-methylated and bound by BMI1, RING2, and SUZ12. Inactivation of pRB family proteins abolishes H3K27 methylation and disrupts BMI1, RING2, and SUZ12 binding to the p16 locus. These results suggest a model in which pRB proteins recruit PRC2 to trimethylate p16, priming the BMI1-containing PRC1L ubiquitin ligase complex to silence p16.
MicroRNAs regulate the expression of the alternative splicing factor nPTB during muscle development
Authors: Boutz Paul L. , Chawla Geetanjali , Stoilov Peter , Black Douglas L.
Published: January 2007

Alternative pre-mRNA splicing determines many changes in gene expression during development. Two regulators known to control splicing patterns during neuron and muscle differentiation are the polypyrimidine tract-binding protein (PTB) and its neuronal homolog nPTB. These proteins repress certain exons in early myoblasts, but upon differentiation of mature myotubes PTB/nPTB expression is reduced, leading to increased inclusion of their target exons. We show here that the repression of nPTB expression during myoblast differentiation results from its targeting by the muscle-restricted microRNA miR-133. During differentiation of C2C12 myoblasts, nPTB protein but not mRNA expression is strongly reduced, concurrent with the up-regulation of miR-133 and the induction of splicing for several PTB-repressed exons. Introduction of synthetic miR-133 into undifferentiated C2C12 cells leads to a decrease in endogenous nPTB expression. Both the miR-133 and the coexpressed miR-1/206 microRNAs are extremely conserved across animal species, and PTB proteins are predicted targets for these miRNAs in Drosophila, mice, and humans. There are two potential miR-133-responsive elements (MRE) within the nPTB 3′ untranslated region (UTR), and a luciferase reporter carrying this 3′ UTR is repressed by miR-133 in an MRE-dependent manner. Transfection of locked nucleic acid (LNA) oligonucleotides designed to block the function of miR-133 and miR-1/206 increases expression of nPTB and decreases the inclusion of PTB dependent exons. These results indicate that miR-133 directly down-regulates a key splicing factor during muscle development and establishes a role for microRNAs in the control of a developmentally dynamic splicing program.
ArabidopsisWEE1 Kinase Controls Cell Cycle Arrest in Response to Activation of the DNA Integrity Checkpoint
Authors: De Schutter Kristof , Joubès Jérôme , Cools Toon , Verkest Aurine , Corellou Florence , Babiychuk Elena , Van Der Schueren Els , Beeckman Tom , Kushnir Sergeï , Inzé Dirk , De Veylder Lieven
Published: January 2007

Upon the incidence of DNA stress, the ataxia telangiectasia–mutated (ATM) and Rad3-related (ATR) signaling kinases activate a transient cell cycle arrest that allows cells to repair DNA before proceeding into mitosis. Although the ATM-ATR pathway is highly conserved over species, the mechanisms by which plant cells stop their cell cycle in response to the loss of genome integrity are unclear. We demonstrate that the cell cycle regulatory WEE1 kinase gene of Arabidopsis thaliana is transcriptionally activated upon the cessation of DNA replication or DNA damage in an ATR- or ATM-dependent manner, respectively. In accordance with a role for WEE1 in DNA stress signaling, WEE1-deficient plants showed no obvious cell division or endoreduplication phenotype when grown under nonstress conditions but were hypersensitive to agents that impair DNA replication. Induced WEE1 expression inhibited plant growth by arresting dividing cells in the G2-phase of the cell cycle. We conclude that the plant WEE1 gene is not rate-limiting for cycle progression under normal growth conditions but is a critical target of the ATR-ATM signaling cascades that inhibit the cell cycle upon activation of the DNA integrity checkpoints, coupling mitosis to DNA repair in cells that suffer DNA damage.
Plasmacytoid dendritic cell leukaemia/lymphoma: towards a well defined entity?
Authors: Garnache‐Ottou Francine , Feuillard Jean , Saas Philippe
Published: January 2007

CD4+/CD56+ haematodermic neoplasm or ‘early’ plasmacytoid dendritic cell leukaemia/lymphoma (pDCL) was described as a disease entity in the last World Health Organisation/European Organisation for Research and Treatment of Cancer classification for cutaneous lymphomas. These leukaemia/lymphomas co‐express CD4 and CD56 without any other lineage‐specific markers and have been identified as arising from plasmacytoid dendritic cells. Despite a fairly homogeneous pattern of markers expressed by most pDCL, numerous distinctive features (e.g. cytological aspects and aberrant marker expression) have been reported. This may be related to the ‘lineage‐independent developmental’ programme of dendritic cells, which may be able to develop from either immature or already committed haematopoietic progenitors. This highlights the need for specific validated markers to diagnose such aggressive leukaemia. Here, we propose –among others (e.g. T‐cell leukaemia 1) – blood dendritic cell antigen‐2 and high levels of CD123 expression as potential markers. In addition, we propose a multidisciplinary approach including several fields of haematology to improve pDCL diagnosis.
Matched sibling donor haematopoietic stem cell transplantation in Fanconi anaemia: an update of the Cincinnati Children's experience
Authors: Farzin Azadeh , Davies Stella M. , Smith Franklin O. , Filipovich Alexandra , Hansen Matthew , Auerbach Arleen D. , Harris Richard E.
Published: January 2007

Our results for 18 patients undergoing matched sibling donor stem cell transplant for Fanconi anaemia at Cincinnati Children's Hospital Medical Center were published in 1994. The present report updates our results in 35 consecutive patients. Thirty patients transplanted for marrow aplasia received cyclophosphamide 5 mg/kg for 4 d and 400 cGy thoraco‐abdominal irradiation. Five patients with clones involving chromosome 7, myelodysplastic syndrome or leukaemia received a more aggressive regimen with total body irradiation. Horse antithymocyte globulin was administered in the pretransplant period to promote engraftment and in the post‐transplant period for additional graft‐versus‐host disease (GVHD) prophylaxis. The median age at bone marrow transplantation was 7·6 years. Median day of engraftment was day +12 (range 9–49), eight patients developed acute GVHD and four chronic GVHD, one limited and three extensive. Twenty‐nine of 35 patients (89% actuarial survival at 10 years) had survived with a median follow up of 10·2 years; two children had developed secondary malignancy. All surviving patients had normal blood counts with full donor engraftment. These data indicate excellent long‐term outcomes and serve as a reference for newer radiation‐free preparative regimes that may reduce the risk of late secondary malignancy.
Upregulation of translational machinery and distinct genetic subgroups characterise hyperdiploidy in multiple myeloma
Authors: Agnelli Luca , Fabris Sonia , Bicciato Silvio , Basso Dario , Baldini Luca , Morabito Fortunato , Verdelli Donata , Todoerti Katia , Lambertenghi‐Deliliers Giorgio , Lombardi Luigia , Neri Antonino
Published: January 2007

Karyotypic instability, including numerical and structural chromosomal aberrations, represents a distinct feature of multiple myeloma (MM). About 40–50% of patients display hyperdiploidy, defined by recurrent trisomies of non‐random chromosomes. To molecularly characterise hyperdiploid (H) and nonhyperdiploid (NH) MM, we analysed the gene expression profiles of 66 primary tumours, and used fluorescence in situ hybridisation to investigate the major chromosomal alterations. The differential expression of 225 genes mainly involved in protein biosynthesis, transcriptional machinery and oxidative phosphorylation distinguished the 28 H‐MM from the 38 NH‐MM cases. The 204 upregulated genes in H‐MM mapped mainly to the chromosomes involved in hyperdiploidy, and the 29% upregulated genes in NH‐MM mapped to 16q. The identified transcriptional fingerprint was robustly validated on a publicly available gene expression dataset of 64 MM cases; and the global expression modulation of regions on the chromosomes involved in hyperdiploidy was verified using a self‐developed non‐parametric statistical method. H‐MM could be further divided into two distinct molecular and transcriptional entities, characterised by the presence of trisomy 11 and 1q‐extracopies/chromosome 13 deletion respectively. These data reinforce the importance of combining molecular cytogenetics and gene expression profiling to define a genomic framework for the study of MM pathogenesis and clinical management.
Bruton's tyrosine kinase prevents activation of the anti‐apoptotic transcription factor STAT3 and promotes apoptosis in neoplastic B‐cells and B‐cell precursors exposed to oxidative stress
Authors: Uckun Fatih , Ozer Zahide , Vassilev Alexei
Published: January 2007

Bruton's tyrosine kinase (BTK) was previously demonstrated to be a mediator of oxidative stress‐induced apoptosis in irradiated neoplastic B‐cells and B‐cell precursors. Defective BTK expression in leukaemic B‐cell precursors from infants with t(4;11) acute lymphoblastic leukaemia has been associated with radiation resistance. The present study examined whether BTK mediates apoptosis during oxidative stress by interfering with the anti‐apoptotic function of signal transducer and activator of transcription 3 (STAT3). BTK physically associated with and tyrosine phosphorylated STAT3; this association was promoted by pervanadate (PV)‐induced oxidative stress. The BTK/STAT3 interaction appeared to prevent STAT3 response to oxidative stress, because PV‐induced STAT3 activation was markedly enhanced in DT40 chicken lymphoma B‐cells that were rendered BTK‐deficient by targeted disruption of the btk gene as well as in BTK‐deficient RAMOS‐1 human lymphoma B‐cells. These BTK‐deficient cells were highly resistant to oxidative stress‐induced apoptosis triggered by PV treatment. Reconstitution of BTK‐deficient DT40 cells with wild‐type human BTK gene eliminated the amplification of the STAT3 response and restored the PV‐induced apoptotic signal. Similarly, while the BTK‐positive NALM‐6 human leukaemic B‐cell precursor cell line showed no STAT3 activation after PV treatment and was exquisitely sensitive to PV‐induced apoptosis, PV failed to induce apoptosis in BTK‐deficient RAMOS‐1 human lymphoma B‐cells that showed a robust STAT3 response. These results provide unprecedented biochemical and genetic evidence for a unique mode of cross‐talk that occurs between BTK and STAT3 pathways during oxidative stress, whereby BTK may trigger apoptosis via negative regulation of the anti‐apoptotic STAT3 activity.
Effects of testosterone and nandrolone on cardiac function: a randomized, placebo‐controlled study
Authors: Chung T. , Kelleher S. , Liu P. Y. , Conway A. J. , Kritharides L. , Handelsman D. J.
Published: January 2007

Background  Androgens have striking effects on skeletal muscle, but the effects on human cardiac muscle function are not well defined, neither has the role of metabolic activation (aromatization, 5α reduction) of testosterone on cardiac muscle been directly studied.

Objective  To assess the effects of testosterone and nandrolone, a non‐amplifiable and non‐aromatizable pure androgen, on cardiac muscle function in healthy young men.

Design  Double‐blind, randomized, placebo‐controlled, three‐arm parallel group clinical trial.

Setting  Ambulatory care research centre.

Participants  Healthy young men randomized into three groups of 10 men.

Intervention  Weekly intramuscular injections of testosterone (200 mg mixed esters), nandrolone (200 mg nandrolone decanoate) or matching (2 ml arachis oil vehicle) placebo for 4 weeks.

Main outcome measures  Comprehensive measures of cardiac muscle function involving transthoracic cardiac echocardiography measuring myocardial tissue velocity, peak systolic strain and strain rates, and bioimpedance measurement of cardiac output and systematic vascular resistance.

Results  Left ventricular (LV) function (LV ejection fraction, LV modified TEI index), right ventricular (RV) function (ejection area, tricuspid annular systolic planar motion, RV modified TEI index) as well as cardiac afterload (mean arterial pressure, systemic vascular resistance) and overall cardiac contractility (stroke volume, cardiac output) were within age‐ and gender‐specific reference ranges and were not significantly (P < 0·05) altered by either androgen or placebo over 4 weeks of treatment. Minor changes remaining within normal range were observed solely within the testosterone group for: increased LV end‐systolic diameter (30 ± 7 vs. 33 ± 5 mm, P = 0·04) and RV end‐systolic area (12·8 ± 1·3 vs. 14·6 ± 3·3 cm2, P = 0·04), reduced LV diastolic septal velocity (Em, 9·5 ± 2·6 vs. 8·7 ± 2·0 cm/s, P = 0·006), increased LV filling pressure (E/Em ratio, 7·1 ± 1·6 vs. 8·3 ± 1·8, P = 0·02) and shortened PR interval on the electrocardiogram (167 ± 13 vs. 154 ± 12, P = 0·03).

Conclusion  Four weeks of treatment with testosterone or nandrolone had no benefical or adverse effects compared with placebo on cardiac function in healthy young men.
Primary testicular failure in Klinefelter's syndrome: the use of bivariate luteinizing hormone‐testosterone reference charts
Authors: Aksglaede Lise , Andersson Anna‐Maria , Jørgensen Niels , Jensen Tina Kold , Carlsen Elisabeth , McLachlan Robert I. , Skakkebæk Niels E. , Petersen Jørgen Holm , Juul Anders
Published: January 2007

Background  The diagnosis of androgen deficiency is based on clinical features and confirmatory low serum testosterone levels. In early primary testicular failure, a rise in serum LH levels suggests inadequate androgen action for the individual's physiological requirements despite a serum testosterone level within the normal range. The combined evaluation of serum LH and testosterone levels in the evaluation of testicular failure has not been widely advocated.

Patients  Seven hundred and six healthy males and 39 patients with known primary hypogonadism due to Klinefelter's syndrome and SRY‐positive 46,XX karyotypes were included in the study.

Design  Testosterone, oestradiol and LH serum concentrations were measured in all individuals. Based on the 706 healthy males two‐dimensional bivariate LH‐testosterone reference charts were constructed.

Results  Despite a median serum total and free testosterone and oestradiol levels being reduced (P < 0·001) and LH levels elevated (P < 0·001) in Klinefelter's syndrome and 46,XX‐males, many subjects (69%) had total testosterone within the reference range. However, using the bivariate charts all subjects lay outside the 97·5 percentile.

Conclusion  Bivariate LH and testosterone charts are useful in the evaluation of men with known primary testicular failure due to sex chromosomal aneuploidy in whom evaluation based on testosterone measurement in isolation underestimates the prevalence of hypogonadism. It is, however, important to emphasize that isolated use of the bivariate evaluation should not form the basis for androgen substitution therapy. Further studies are needed in order to evaluate the use of bivariate LH and testosterone charts in the assessment of any younger man with possible primary testicular failure.
Serum levels of adiponectin and IGFBP‐1 in short children born small for gestational age
Authors: Kamoda Tomohiro , Nozue Hiroki , Matsui Akira
Published: January 2007

Objective  The aim of this study was to quantify serum adiponectin concentrations in short children born small for gestational age (SGA) compared with those in children born appropriate for gestational age (AGA), and to assess the relationship between the serum levels of adiponectin and insulin‐like growth factor binding protein‐1 (IGFBP‐1) known as a predictor of the development of type 2 diabetes mellitus and cardiovascular disease.

Subjects and methods  Sixteen prepubertal short children born SGA and 20 short children born AGA, matched for age, body mass index, height, pubertal status, gestational age, bone age and midparental height, were included in the study. The serum levels of adiponectin, IGFBP‐1, insulin and insulin‐like growth factor‐I (IGF‐I) were measured in the fasting state.

Results  The levels of serum adiponectin were significantly lower in the SGA than in AGA children (10·5 ± 4·2 vs. 13·9 ± 5·1 µg/ml, P < 0·05). The levels of serum IGFBP‐1, insulin and IGF‐I were all similar in both groups. Overall, there was a significant positive correlation between adiponectin and IGFBP‐1 (r = 0·40, P < 0·05).

Conclusions  Our results suggest that hypoadiponectinaemia in short SGA children without catch‐up growth may reflect insulin resistance and imply a higher risk of developing type 2 diabetes mellitus. Additionally, adiponectin may be a more sensitive indicator for latent insulin resistance than IGFBP‐1 in short SGA children.
Growth hormone distribution kinetics are markedly reduced in adults with growth hormone deficiency
Authors: Catalina Pablo F. , Páramo Concepción , Andrade Maria Amalia , Mallo Federico
Published: January 2007

Objective  Growth hormone (GH) circulating levels are highly dependent not only on GH secretion rate from the pituitary, but also on the hormone distribution in the compartments of the body and elimination phenomena. In adult GH‐deficient patients these factors become critical nowadays, especially when recombinant human GH (rhGH) is available for replacement therapy. In the present study, we assess the influence of both distribution and elimination phenomena on GH pharmacokinetics in adult GH‐deficient patients.

Methods  We used a four‐step methodology following a compartmental approach after an intravenous bolus of recombinant GH in adult GH‐deficient patients.

Results  We found that GH kinetics are clearly explained by a bi‐exponential, two‐compartmental model in GH‐deficient patients, similarly than in normal or diabetic subjects, as previously shown. We have also observed a marked delay in the whole GH elimination process in GH‐deficient patients compared to normal adult subjects, as revealed by metabolic clearance ratio (MCR), elimination constant from central compartment (k10), and mean resident time in the body (MRT). Interestingly, such a delay appear to be caused by deep changes in the distribution phase (Mtt1– mean transit time‐1; T1/2α– GH half‐life at distribution phase), while the elimination phenomenon remains unaltered.

Conclusion  Our results emphasize the relevance of distribution phenomena in GH pharmacokinetics, and indicates that studies avoiding data from the GH distribution phase, such as those carried out in steady‐state conditions, or those using noncompartmental models, could easily miss relevant information. Our data should be taken into consideration when establishing the appropriate dosage for GH replacement treatments in GH‐deficient patients, and calculations should include GH distribution kinetics.
Temperature and soil moisture effects on dissolved organic matter release from a moorland Podzol O horizon under field and controlled laboratory conditions
Authors: Stutter M. I. , Lumsdon D. G. , Cooper R. J.
Published: January 2007

Organic upland soils store large amounts of humified organic matter. The mechanisms controlling the leaching of this C pool are not completely understood. To examine the effects of temperature and microbial cycling on C leaching, we incubated five unvegetated soil cores from a Podzol O horizon (from NE Scotland), over a simulated natural temperature cycle for 1 year, whilst maintaining a constant soil moisture content. Soil cores were leached with artificial rain (177 mm each, monthly) and the leachates analysed for dissolved organic carbon (DOC) and their specific C‐normalized UV absorbance determined (SUVA, 285 nm). Monthly values of respiration of the incubated soils were determined as CO2 efflux. To examine the effects of vegetation C inputs and soil moisture, in addition to temperature, we sampled O horizon pore waters in situ and collected five additional field soil cores every month. The field cores were leached under controlled laboratory conditions. Hysteresis in the monthly amount of DOC leached from field cores resulted in greater DOC on the rising, than falling temperature phases. This hysteresis suggested that photosynthetic C stimulated greater DOC losses in early summer, whereas limitations in the availability of soil moisture in late summer suppressed microbial decomposition and DOC loss. Greater DOC concentrations of in‐situ pore waters than for any core leachates were attributed to the effects of soil drying and physico‐chemical processes in the field. Variation in the respiration rates for the incubated soils was related to temperature, and respiration provided a greater pathway of C loss (44 g C m−2 year−1) than DOC (7.2 g C m−2 year−1). Changes in SUVA over spring and summer observed in all experimental systems were related to the period of increased temperature. During this time, DOC became less aromatic, which suggests that lower molecular weight labile compounds were not completely mineralized. The ultimate DOC source appears to be the incomplete microbial decomposition of recalcitrant humified C. In warmer periods, any labile C that is not respired is leached, but in autumn either labile C production ceases, or it is sequestered in soil biomass.
Iron speciation in soils and soil aggregates by synchrotron‐based X‐ray microspectroscopy (XANES,μ‐XANES)
Authors: Prietzel J. , Thieme J. , Eusterhues K. , Eichert D.
Published: January 2007

Iron speciation in soils is still poorly understood. We have investigated inorganic and organic standard substances, diluted mixtures of common Fe minerals in soils (pyrite, ferrihydrite, goethite), soils in a forested watershed which constitute a toposequence with a hydrological gradient (Dystric Cambisol, Dystric Planosol, Rheic Histosol), and microsites of a dissected soil aggregate by X‐ray Absorption Near Edge Spectroscopy (XANES) at the iron K‐edge (7112 eV) to identify different Fe(II) and Fe(III) components. We calculated the pre‐edge peak centroid energy of all spectra and quantified the contribution of different organic and inorganic Fe‐bearing compounds by Linear Combination Fitting (LCF) conducted on the entire spectrum (E = 7085–7240 eV) and on the pre‐edge peak. Fe‐XANES conducted on organic and inorganic standards and on synthetic mixtures of pyrite, ferrihydrite and goethite showed that by calculating the pre‐edge peak centroid energy, the Fe(II)/Fe(III) ratio of different Fe‐bearing minerals (Fe sulphides, Fe oxyhydroxides) in mineral mixtures and soils can be quantified with reasonable accuracy. A more accurate quantification of the Fe(II)/Fe(III) ratio was possible with LCF conducted on the entire XANES spectrum. For the soil toposequence, an increased groundwater influence from the Cambisol to the Histosol was reflected in a larger contribution of Fe(II) compounds (Fe(II) silicate, Fe monosulphide, pyrite) and a smaller contribution of Fe(III) oxyhydroxides (ferrihydrite, goethite) to total iron both in the topsoil and the subsoil. In the organic topsoils, organically bonded Fe (33–45% of total Fe) was 100% Fe(III). For different microsites in the dissected aggregate, spatial resolution ofμ‐XANES revealed different proportions of Fe(II) and Fe(III) compounds. Fe K‐edge XANES andμ‐XANES allows an approximate quantification of Fe(II) and Fe(III) and different Fe compounds in soils and (sub)micron regions of soil sections, such as mottles, concretions, and rhizosphere regions, thus opening new perspectives in soil research.
Allocation and residence time of photosynthetic products in a boreal forest using a low‐level 14C pulse‐chase labeling technique
Authors: CARBONE MARIAH S. , CZIMCZIK CLAUDIA I. , McDUFFEE KELSEY E. , TRUMBORE SUSAN E.
Published: January 2007

Much of our understanding about how carbon (C) is allocated in plants comes from radiocarbon (14C) pulse‐chase labeling experiments. However, the large amounts of 14C required for decay‐counting mean that these studies have been restricted for the most part to mesocosm or controlled laboratory experiments. Using the enhanced sensitivity for 14C detection available with accelerator mass spectrometry (AMS), we tested the utility of a low‐level 14C pulse‐chase labeling technique for quantifying C allocation patterns and the contributions of different plant components to total ecosystem respiration in a black spruce forest stand in central Manitoba, Canada. All aspects of the field experiment used 14C at levels well below regulated health standards, without significantly altering atmospheric CO2 concentrations. Over 30 days following the label application in late summer (August and September), we monitored the temporal and spatial allocation patterns of labeled photosynthetic products by measuring the amount and 14C content of CO2 respired from different ecosystem components. The mean residence times (MRT) for labeled photosynthetic products to be respired in the understory (feather mosses), canopy (black spruce), and rhizosphere (black spruce roots and associated microbes) were <1, 6, and 15 days, respectively. Respiration from the canopy and understory showed significantly greater influence of labeled photosynthates than excised root and intact rhizosphere respiration. After 30 days,∼65% of the label assimilated had been respired by the canopy,∼20% by the rhizosphere, and∼9% by the understory, with∼6% unaccounted for and perhaps remaining in tissues. Maximum 14C values in root and rhizosphere respiration were reached 4 days after label application. The label was still detectable in root, rhizosphere and canopy respiration after 30 days; these levels of remaining label would not have been detectible had a 13C label been applied. Our results support previous studies indicating that a substantial portion of the C fueling rhizosphere respiration in the growing season may be derived from stored C pools rather than recent photosynthetic products.
Elevated CO2 increases microbial carbon substrate use and nitrogen cycling in Mojave Desert soils
Authors: JIN V. L. , EVANS R. D.
Published: January 2007

Identifying soil microbial responses to anthropogenically driven environmental changes is critically important as concerns intensify over the potential degradation of ecosystem function. We assessed the effects of elevated atmospheric CO2 on microbial carbon (C) and nitrogen (N) cycling in Mojave Desert soils using extracellular enzyme activities (EEAs), community‐level physiological profiles (CLPPs), and gross N transformation rates. Soils were collected from unvegetated interspaces between plants and under the dominant shrub (Larrea tridentata) during the 2004–2005 growing season, an above‐average rainfall year. Because most measured variables responded strongly to soil water availability, all significant effects of soil water content were used as covariates to remove potential confounding effects of water availability on microbial responses to experimental treatment effects of cover type, CO2, and sampling date. Microbial C and N activities were lower in interspace soils compared with soils under Larrea, and responses to date and CO2 treatments were cover specific. Over the growing season, EEAs involved in cellulose (cellobiohydrolase) and orthophosphate (alkaline phosphatase) degradation decreased under ambient CO2, but increased under elevated CO2. Microbial C use and substrate use diversity in CLPPs decreased over time, and elevated CO2 positively affected both. Elevated CO2 also altered microbial C use patterns, suggesting changes in the quantity and/or quality of soil C inputs. In contrast, microbial biomass N was higher in interspace soils than soils under Larrea, and was lower in soils exposed to elevated CO2. Gross rates of NH4+ transformations increased over the growing season, and late‐season NH4+ fluxes were negatively affected by elevated CO2. Gross NO3− fluxes decreased over time, with early season interspace soils positively affected by elevated CO2. General increases in microbial activities under elevated CO2 are likely attributable to greater microbial biomass in interspace soils, and to increased microbial turnover rates and/or metabolic levels rather than pool size in soils under Larrea. Because soil water content and plant cover type dominates microbial C and N responses to CO2, the ability of desert landscapes to mitigate or intensify the impacts of global change will ultimately depend on how changes in precipitation and increasing atmospheric CO2 shift the spatial distribution of Mojave Desert plant communities.
Development and characterization of novel photosensitizer :  scFv conjugates for use in photodynamic therapy of cancer
Authors: Staneloudi Chrysovalanto , Smith Karen A. , Hudson Robert , Malatesti Nela , Savoie Huguette , Boyle Ross W. , Greenman John
Published: January 2007

Photodynamic therapy (PDT) is becoming an evermore useful tool in oncology but is frequently limited by side‐effects caused by a lack of targeting of the photosensitizer. This problem can often be circumvented by the conjugation of photosensitizers to tumour‐specific monoclonal antibodies. An alternative is the use of single chain (sc) Fv fragments which, whilst retaining the same binding specificity, are more efficient at penetrating tumour masses because of their smaller size; and are more effectively cleared from the circulation because of the lack of an Fc domain. Here we describe the conjugation of two isothiocyanato porphyrins to colorectal tumour‐specific scFv, derived from an antibody phage display library. The conjugation procedure was successfully optimized and the resulting immunoconjugates showed no loss of cell binding. In vitro assays against colorectal cell lines showed these conjugates had a selective photocytotoxic effect on cells. Annexin V and propidium iodide staining of treated cells confirmed cell death was mediated principally via an apoptotic pathway. This work suggests that scFv : porphyrin conjugates prepared using isothiocyanato porphyrins show promise for use as targeted PDT agents.
Evolution of sex chromosomes: dosage compensation of the Lcp1–4 gene cluster on the evolving neo‐X chromosome in Drosophila miranda
Authors: Steinemann S. , Steinemann M.
Published: January 2007

In Drosophila miranda the small multigene family of the larval cuticle protein (Lcp1–4) genes resides on the evolving neo‐X and neo‐Y sex chromosome pair while in the sibling species Drosophila pseudoobscura and Drosophila persimilis the gene cluster is inherited autosomally. The neo‐Y chromosomal Lcp1, Lcp2 and Lcp4 genes are, as previously shown by us, not expressed and only Lcp3 is expressed at a strongly reduced level. As a first step in understanding the evolutionary mechanism(s) transforming an autosome into a dosage compensated X we analysed the expression behaviour and promoter structure of the Lcp1–4 genes on the neo‐X. The normalized relative expression levels reveal that all four neo‐X chromosomal Lcp genes in D. miranda males, including Lcp3, are already dosage compensated.
Effective Use of Microarrays in Neuroendocrine Research
Authors: Chu T. T. , Fink M. Y. , Mong J. A. , John G. , Auger A. P. , Ge Y. , Sealfon S. C.
Published: January 2007

The development of microarray technology makes it possible to simultaneously assay the expression level of hundreds to tens of thousands of mRNA transcripts in one experiment. Genome‐wide transcriptional analysis has increasing importance for many areas of neuroendocrinology research. The expense and technical complexity of microarray experiments can make it difficult to navigate the terrain of rival platforms and technologies. In this review, we provide a practical view and comparison of various microarray technologies. Affymetrix arrays, high‐density cDNA arrays, membrane arrays and experimental design and data analysis are all discussed by researchers currently using these techniques to study gene regulation in neuroendocrine tissues.
Thyrotrophin‐Releasing Hormone Decreases Feeding and Increases Body Temperature, Activity and Oxygen Consumption in Siberian Hamsters
Authors: Schuhler S. , Warner A. , Finney N. , Bennett G. W. , Ebling F. J. P. , Brameld J. M.
Published: January 2007

Thyrotrophin‐releasing hormone (TRH) is known to play an important role in the control of food intake and energy metabolism in addition to its actions on the pituitary‐thyroid axis. We have previously shown that central administration of TRH decreases food intake in Siberian hamsters. This species is being increasingly used as a physiological rodent model in which to understand hypothalamic control of long‐term changes in energy balance because it accumulates fat reserves in long summer photoperiods, and decreases food intake and body weight when exposed to short winter photoperiods. The objectives of our study in Siberian hamsters were: (i) to investigate whether peripheral administration of TRH would mimic the effects of central administration of TRH on food intake and whether these effects would differ dependent upon the ambient photoperiod; (ii) to determine whether TRH would have an effect on energy expenditure; and (iii) to investigate the potential sites of action of TRH. Both peripheral (5–50 mg/kg body weight; i.p.) and central (0.5 µg/ml; i.c.v.) administration of TRH decreased food intake, and increased locomotor activity, body temperature and oxygen consumption in the Siberian hamster, with a rapid onset and short duration of action. Systemic treatment with TRH was equally effective in suppressing feeding regardless of ambient photoperiod. The acute effects of TRH are likely to be centrally mediated and independent of its role in the control of the production of thyroid hormones. We conclude that TRH functions to promote a catabolic energetic state by co‐ordinating acute central and chronic peripheral (thyroid‐mediated) function.
Interaction Between Oestrogen and Oxytocin on Hypothalamic‐Pituitary‐Adrenal Axis Activity
Authors: Ochedalski T. , Subburaju S. , Wynn P. C. , Aguilera G.
Published: January 2007

In addition to its role in reproduction, oxytocin has central actions modulating behavioural and hypothalamic‐pituitary‐adrenal (HPA) axis responses during late pregnancy and lactation. The hypothesis that ovarian hormones modulate the effects of oxytocin on HPA axis activity was studied in 7‐day ovariectomised rats receiving oestradiol with or without progesterone replacement and intracerebroventricular (i.c.v) minipump infusion of oxytocin (100 ng/h). In an initial experiment, i.c.v. oxytocin had no effect on basal or restraint‐stimulated plasma adrenocorticotrophic hormone (ACTH) and corticosterone concentrations or hypothalamic corticotrophin‐releasing factor (CRF) mRNA expression with low oestradiol replacement alone but it had a stimulatory effect in the presence of low oestradiol and progesterone. To investigate further whether oestradiol modulates central actions of oxytocin, rats received low dioestrous (low), pro‐oestrous (medium) or pregnancy (high) oestradiol replacement levels, yielding plasma concentrations of < 5, 17.3 ± 4.5 and 258 ± 32 pg/ml, respectively, with or without i.c.v. oxytocin. Oestradiol caused dose‐dependent increases in basal plasma ACTH and corticosterone concentrations but decreased the ACTH response to restraint stress. In parallel to the changes in basal plasma ACTH, high oestrogen increased basal CRF hnRNA, CRF mRNA in the paraventricular nucleus and pro‐opiomelanocortin (POMC) mRNA in the pituitary gland, while decreasing restraint stress‐stimulated levels. Intracerebroventricular administration of oxytocin reduced basal and stress‐stimulated plasma ACTH, hypothalamic CRF hnRNA (30 min), CRF mRNA and pituitary POMC mRNA (4 h) levels parallel to the increases induced by elevating plasma oestradiol. The present study demonstrates the converse effects of oestradiol on basal and restraint stress‐stimulated basal HPA axis activity, and that the ability of central oxytocin to inhibit HPA axis activity depends on the levels of circulating oestradiol.
An α‐amylase (At4g25000) in Arabidopsis leaves is secreted and induced by biotic and abiotic stress
Authors: DOYLE ELIZABETH A. , LANE ASHLEIGH M. , SIDES JAMES M. , MUDGETT MARY BETH , MONROE JONATHAN D.
Published: January 2007

Leaves are reported to contain a secreted α‐amylase that accumulates during senescence or after biotic or abiotic stress; however, a gene encoding this enzyme has not been described. Because a secreted amylase is isolated from plastidic starch, the function of this enzyme is difficult to predict, but circumstantial evidence suggests that it may degrade starch after cell death. The Arabidopsis thaliana genome contains three α‐amylase genes, one of which, AMY1 (At4g25000), has a putative signal sequence suggesting that the protein may be secreted. Two independent T‐DNA insertion mutants in AMY1 lacked an amylase band on starch zymograms, which was previously named ‘A1’. Washed leaf protoplasts contained reduced A1 activity suggesting that the enzyme is secreted. Native AMY1, fused to a weakly fluorescent form of GFP, was sensitive to proteinase K infiltrated into leaf apoplastic spaces, while a cytosolic form of GFP was unaffected until cell breakage, confirming that the AMY1 protein is secreted. Amylase A1 was transcriptionally induced in senescing leaves and in leaves exposed to heat stress, treated with abscisic acid or infected with Pseudomonas syringae pv. tomato expressing avrRpm1. The A1 amylase was also extremely heat resistant and its expression was up‐regulated in cpr5‐2, an activated defence response mutant.
Proteomic analysis of leaf proteins during dehydration of the resurrection plant Xerophyta viscosa
Authors: INGLE ROBERT A. , SCHMIDT ULRIKE G. , FARRANT JILL M. , THOMSON JENNIFER A. , MUNDREE SAGADEVAN G.
Published: January 2007

The desiccation‐tolerant phenotype of angiosperm resurrection plants is thought to rely on the induction of protective mechanisms that maintain cellular integrity during water loss. Two‐dimensional (2D) sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of the Xerophyta viscosa Baker proteome was carried out during dehydration to identify proteins that may play a role in such mechanisms. Quantitative analysis revealed a greater number of changes in protein expression levels at 35% than at 65% relative water content (RWC) compared to fully hydrated plants, and 17 dehydration‐responsive proteins were identified by tandem mass spectrometry (MS). Proteins showing increased abundance during drying included an RNA‐binding protein, chloroplast FtsH protease, glycolytic enzymes and antioxidants. A number of photosynthetic proteins declined sharply in abundance in X. viscosa at RWC below 65%, including four components of photosystem II (PSII), and Western blot analysis confirmed that two of these (psbP and Lhcb2) were not detectable at 30% RWC. These data confirm that poikilochlorophylly in X. viscosa involves the breakdown of photosynthetic proteins during dismantling of the thylakoid membranes. In contrast, levels of these photosynthetic proteins were largely maintained during dehydration in the homoiochlorophyllous species Craterostigma plantagineum Hochst, which does not dismantle thylakoid membranes on drying.
The Arabidopsis thaliana sulfiredoxin is a plastidic cysteine‐sulfinic acid reductase involved in the photooxidative stress response
Authors: Rey Pascal , Bécuwe Noëlle , Barrault Marie‐Bénédicte , Rumeau Dominique , Havaux Michel , Biteau Benoît , Toledano Michel B.
Published: January 2007

The 2‐cysteine peroxiredoxins (2‐Cys‐Prxs) are antioxidants that reduce peroxides through a thiol‐based mechanism. During catalysis, these ubiquitous enzymes are occasionally inactivated by the substrate‐dependent oxidation of the catalytic cysteine to the sulfinic acid (‐SO2H) form, and are reactivated by reduction by sulfiredoxin (Srx), an enzyme recently identified in yeast and in mammal cells. In plants, 2‐Cys‐Prxs constitute the most abundant Prxs and are located in chloroplasts. Here we have characterized the unique Srx gene in Arabidopsis thaliana (AtSrx) from a functional point of view, and analyzed the phenotype of two AtSrx knockout (AtSrx–) mutant lines. AtSrx is a chloroplastic enzyme displaying sulfinic acid reductase activity, as shown by the ability of the recombinant AtSrx to reduce the overoxidized 2‐Cys‐Prx form in vitro, and by the accumulation of the overoxidized Prx in mutant lines lacking Srx in vivo. Furthermore, AtSrx mutants exhibit an increased tolerance to photooxidative stress generated by high light combined with low temperature. These data establish that, as in yeast and in mammals, plant 2‐Cys‐Prxs are subject to substrate‐mediated inactivation reversed by Srx, and suggest that the 2‐Cys‐Prx redox status and sulfiredoxin are parts of a signaling mechanism participating in plant responses to oxidative stress.
HY5 is a point of convergence between cryptochrome and cytokinin signalling pathways in Arabidopsis thaliana
Authors: Vandenbussche Filip , Habricot Yvette , Condiff Amanda S. , Maldiney Régis , Straeten Dominique Van Der , Ahmad Margaret
Published: January 2007

Blue‐light‐dependent photomorphogenesis in Arabidopsis is regulated principally by the cryptochrome flavin‐type photoreceptors, which control hypocotyl growth inhibition, cotyledon and leaf expansion, and the expression of light‐regulated genes. Interestingly the plant hormone cytokinin induces similar responses when added exogenously to germinating seedlings, suggesting a link between cryptochrome and cytokinin signalling pathways. In this work we explore the relationship between cryptochrome and cytokinin signalling pathways in the promotion of photomorphogenesis. The effect of exogenously added cytokinins on hypocotyl growth inhibition occurs in the dark, and is largely independent and additive to that of cryptochromes in blue light, via distinct signalling pathways. By contrast, cytokinin‐dependent stimulation of anthocyanin accumulation occurs only in light, and interacts with the signalling pathway downstream of cryptochrome 1 (CRY1) at the level of transcript accumulation of anthocyanin biosynthetic genes. Mutants in elongated hypocotyl 5 (hy5), a downstream intermediate in the CRY1 signalling pathway, show a reduced induction of anthocyanin accumulation in blue light by cytokinins, similar to that observed for cryptochrome (cry1) mutants. Furthermore cytokinins are shown to increase levels of HY5 protein accumulation, suggesting that cytokinins may function by reducing HY5 degradation by COP1 (constitutively photomorphogenic 1). As both cryptochrome and cytokinin signalling pathways increase HY5 protein levels, and as HY5 binds to the promoters of anthocyanin biosynthetic enzymes to stimulate gene expression, it is concluded that the regulation of HY5 protein stability represents a point of convergence between cryptochrome and cytokinin signalling pathways.
Dynamics of MONOPTEROS and PIN‐FORMED1 expression during leaf vein pattern formation in Arabidopsis thaliana
Authors: Wenzel Carol L. , Schuetz Mathias , Yu Qian , Mattsson Jim
Published: January 2007

Genetic evidence links the Arabidopsis MONOPTEROS (MP) and PIN‐FORMED1 (PIN1) genes to the patterning of leaf veins. To elucidate their potential functions and interactions in this process, we have assessed the dynamics of MP and PIN1 expression during vascular patterning in Arabidopsis leaf primordia. Both genes undergo a dynamic process of gradual refinement of expression into files one to two cells wide before overt vascular differentiation. The subcellular distribution of PIN1 is also gradually refined from a non‐polar distribution in isodiametric cells to strongly polarized in elongated procambial cells and provides an indication of overall directions of auxin flow. We found evidence that MP expression can be activated by auxin exposure and that PIN1 as well as DR5::GUS expression is defective in mp mutant leaves. Taken together the results suggest a feedback regulatory loop that involves auxin, MP and PIN1 and provide novel experimental support for the canalization‐of‐auxin‐flow hypothesis.
The ubiquitin pathway is required for innate immunity in Arabidopsis
Authors: Goritschnig Sandra , Zhang Yuelin , Li Xin
Published: January 2007

Plant defences require a multitude of tightly regulated resistance responses. In Arabidopsis, the unique gain‐of‐function mutant suppressor of npr1‐1 constitutive 1 (snc1) carries a point mutation in a Resistance (R)‐gene, resulting in constitutive activation of defence responses without interaction with pathogens. This has allowed us to identify various downstream signalling components essential in multiple defence pathways. One mutant that suppresses snc1‐mediated constitutive resistance is modifier of snc1 5 (mos5), which carries a 15‐bp deletion in UBA1, one of two ubiquitin‐activating enzyme genes in Arabidopsis. A mutation in UBA2 does not suppress snc1, suggesting that these two genes are not equally required in Arabidopsis disease resistance. On the other hand, a mos5 uba2 double mutant is lethal, implying partial redundancy of the two homologues. Apart from affecting snc1‐mediated resistance, mos5 also exhibits enhanced disease susceptibility to a virulent pathogen and is impaired in response to infection with avirulent bacteria carrying the protease elicitor AvrRpt2. The mos5 mutation in the C‐terminus of UBA1 might affect binding affinity of the downstream ubiquitin‐conjugating enzymes, thus perturbing ubiquitination of target proteins. Furthermore, SGT1b and RAR1, which are necessary for resistance conferred by the SNC1‐related R‐genes RPP4 and RPP5, are dispensable in snc1‐mediated resistance. Our data reveal the definite requirement for the ubiquitination pathway in the activation and downstream signalling of several R‐proteins.