Various experimental data indicate signalling roles for nitric oxide (NO) in processes such as xylogenesis, programmed cell death, pathogen defence, flowering, stomatal closure, and gravitropism. ...However, it still remains unclear how NO is synthesized. Nitric oxide synthase-like activity has been measured in various plant extracts, NO can be generated from nitrite via nitrate reductase and other mechanisms of NO generation are also likely to exist. NO removal mechanisms, for example, by reaction with haemoglobins, have also been identified. NO is a gas emitted by plants, with the rate of evolution increasing under conditions such as pathogen challenge or hypoxia. However, exactly how NO evolution relates to its bioactivity in planta remains to be established. NO has both aqueous and lipid solubility, but is relatively reactive and easily oxidized to other nitrogen oxides. It reacts with superoxide to form peroxynitrite, with other cellular components such as transition metals and haem-containing proteins and with thiol groups to form S-nitrosothiols. Thus, diffusion of NO within the plant may be relatively restricted and there might exist 'NO hot-spots' depending on the sites of NO generation and the local biochemical micro-environment. Alternatively, it is possible that NO is transported as chemical precursors such as nitrite or as nitrosothiols that might function as NO reservoirs. Cellular perception of NO may occur through its reaction with biologically active molecules that could function as 'NO-sensors'. These might include either haem-containing proteins such as guanylyl cyclase which generates the second messenger cGMP or other proteins containing exposed reactive thiol groups. Protein S-nitrosylation alters protein conformation, is reversible and thus, is likely to be of biological significance.
Unfractionated peripheral blood stem cell (PBSC) grafts contain measurable quantities of myeloma cells and are therefore a potential source of relapse posttransplantation. In contrast, ...fluorescence-activated cell sorting (FACS)-sorted CD34+Thy1+ Lin− peripheral blood cells are substantially enriched for stem cell activity, yet contain virtually no clonal myeloma cells. A study was performed in patients with symptomatic myeloma, who had received 12 months or less of preceding standard chemotherapy, to evaluate the feasibility of large scale purification of primitive hematopoietic stem cells in order to study engraftment kinetics posttransplantation and the degree of tumor cell contamination of this cell population, based on polymerase chain reaction (PCR) analysis for the patient-specific complementarity-determining region III (CDR III). PBSC were mobilized with high dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF). A combination of elutriation and chemical lysis was used to deplete PBSC collections of monocytes, granulocytes, erythrocytes, and platelets. Subsequently, CD34+ Thy1+ Lin−progenitor cells were purified with high speed cell sorting. Of the 10 evaluable patients, nine met the required minimum criteria of ≥7.2 × 105 cells/kg to support tandem transplants. After high dose melphalan (200 mg/m2) eight engrafted successfully, although granulocyte (absolute neutrophil count ANC >0.5 × 109/L, 16 days) and platelet recovery (platelets > 50 × 109/L, 39 days) was substantially delayed when compared with unmanipulated PBSC grafts; one patient required infusion of a reserve graft because of lack of evidence of engraftment by day +28. Three patients proceeded to a second graft with high dose melphalan and total body irradiation; two required infusion of a reserve graft and both died of infectious complications; one showed delayed, but complete, engraftment after this myeloablative regimen. Two of the nine evaluable patients attained a clinical complete remission (CR). The grafts from three patients were tested for tumor contamination and contained no detectable clonal myeloma cells. Larger quantities of purified cells may be required to resolve the problem of delayed engraftment.
Multiple myeloma (MM) is usually characterized by production of a single serum monoclonal protein of constant isotype and light-chain restriction. Multiple Ig isotypes and isotype switches, which are ...rare in untreated patients, are reported to be more common in patients undergoing myeloablative therapy. These additional protein bands, detected by immunofixation electrophoresis (IFE), could be due to altered paraprotein production by the malignant plasma cell clone or oligoclonal Ig production during recovery of B-cell function after myeloablative therapy. We analyzed abnormal protein bands (APB), distinct from the presenting paraprotein, in 550 patients receiving high-dose therapy with autologous hematopoietic cell transplantation at a single institution. Fifty-five patients (10%) had APB, 48 had oligoclonal bands (OB), and 23 had an apparent isotype switch (IS) on IFE (16 had both OB and IS). Morphologic and flow cytometric examination of bone marrow in 17 patients with IS showed no evidence of a clonal plasma cell isotype switch. Patients with APB had significantly higher complete response to therapy (67% v 37%, P = .001). To assess the independent prognostic relevance of APB, a multivariate analysis was performed among 471 patients surviving at least 12 months from first transplant (all patients developing APB had done so by 12 months from first transplant). APB (in 50 patients) was a favorable feature for both event-free (rank 3, P = .004) and overall survival (rank 3, P = .0005). We propose that OB and IS are likely to be due to recovery of Ig production rather than alterations in the biology of the malignant plasma cell clone.
Haematopoietic growth factors, especially granulocyte colony‐stimulating factor (G‐CSF), are frequently utilized alone for peripheral blood stem cell (PBSC) procurement to avoid the morbidity ...associated with high‐dose chemotherapy (HDT). Moreover, the cytotoxic agents used may not be the most optimal therapy for the malignancy. It also makes scheduling of apheresis easier. Factors having an impact on PBSC procurement and engraftment after HDT were analysed in 117 multiple myeloma patients mobilized with G‐CSF (10–16 µg/kg, median 12 µg/kg) alone using Cox regression analysis. A median of 6·2 × 106 CD34 cells/kg (range 0·6–34·1) were procured during leukapheresis and a median of 2·5 × 106 CD34 cells was infused after the first HDT (range 0·3–23·9). The only factor significantly affecting optimal PBSC procurement was duration of preceding conventional chemotherapy (P = 0·002). Granulocyte recovery was prompt in almost all patients, 75% of whom attained a granulocyte count of 0·5 × 109/l by day 13 (median 11, range 7–19). However, platelet recovery to both 20 × 109/l (median 12 d, range 8–50+) and 50 × 109/l (median 20 d, range 7–205+) varied widely. On univariate analysis, factors influencing platelet recovery were the number of CD34 cells/kg infused, age, β2‐microglobulin levels, response to preceding therapy, bone marrow plasmacytosis and duration of prior therapy. Factors attaining significance on multivariate analysis included number of CD34 cells/kg infused (P = 0·007), β2‐microglobulin levels (P = 0·0001), most probably representing disease load, and age (P = 0·002). Patients with high tumour burden, i.e. β2‐microglobulin levels > 2·5 mg/l, probably benefit from chemotherapy for mobilization both in terms of cytoreduction and adequate stem cell mobilization resulting in accelerated engraftment.
Treatment of Arabidopsis thaliana suspension cultures with H2O2 results in the activation of a 44 kDa protein kinase with the characteristics of a mitogen activated protein (MAP) kinase. It ...preferentially phosphorylates myelin basic protein as an artificial substrate, it requires tyrosine phosphorylation for its activity, is tyrosine and threonine phosphorylated upon activation, and its activation is rapid, transient, and occurs in a dose-dependent manner. This is the first demonstration of the activation of a MAP kinase-like enzyme by H2O2 in plants.
It is now clear that hydrogen peroxide (H2O2) and nitric oxide (NO) function as signalling molecules in plants. A wide range of abiotic and biotic stresses results in H2O2 generation, from a variety ...of sources. H2O2 is removed from cells via a number of antioxidant mechanisms, both enzymatic and non‐enzymatic. Both biotic and abiotic stresses can induce NO synthesis, but the biosynthetic origins of NO in plants have not yet been resolved. Cellular responses to H2O2 and NO are complex, with considerable cross‐talk between responses to several stimuli. In this review the potential roles of H2O2 and NO during various stresses and the signalling pathways they activate are discussed. Key signalling components that might provide targets for enhancing crop production are also identified.
We measure the experimental error that arises from the use of non-validated simulators in computer architecture research, with the goal of increasing the rigor of simulation- based studies. We ...describe the methodology that we used to validate a microprocessor simulator against a Compaq DS-10L workstation, which contains an Alpha 21264 processor. Our evaluation suite consists of a set of 21 microbenchmarks that stress different aspects of the 21264 microarchitecture. Using the microbenchmark suite as the set of workloads, we describe how we reduced our simulator error to an arithmetic mean of 2%, and include details about the specific aspects of the pipeline that required extra care to reduce the error. We show how these low-level optimizations reduce average error from 40% to less than 20% on macrobenchmarks drawn from the SPEC2000 suite. Finally, we examine the degree to which performance optimizations are stable across different simulators, showing that researchers would draw different conclusions, in some cases, if using validated simulators.
Alzheimer's disease (AD) is highly heritable and recent studies have identified over 20 disease-associated genomic loci. Yet these only explain a small proportion of the genetic variance, indicating ...that undiscovered loci remain. Here, we performed a large genome-wide association study of clinically diagnosed AD and AD-by-proxy (71,880 cases, 383,378 controls). AD-by-proxy, based on parental diagnoses, showed strong genetic correlation with AD (r
= 0.81). Meta-analysis identified 29 risk loci, implicating 215 potential causative genes. Associated genes are strongly expressed in immune-related tissues and cell types (spleen, liver, and microglia). Gene-set analyses indicate biological mechanisms involved in lipid-related processes and degradation of amyloid precursor proteins. We show strong genetic correlations with multiple health-related outcomes, and Mendelian randomization results suggest a protective effect of cognitive ability on AD risk. These results are a step forward in identifying the genetic factors that contribute to AD risk and add novel insights into the neurobiology of AD.
Several lines of evidence suggest that genome-wide association studies (GWASs) have the potential to explain more of the “missing heritability” of common complex phenotypes. However, reliable methods ...for identifying a larger proportion of SNPs are currently lacking. Here, we present a genetic-pleiotropy-informed method for improving gene discovery with the use of GWAS summary-statistics data. We applied this methodology to identify additional loci associated with schizophrenia (SCZ), a highly heritable disorder with significant missing heritability. Epidemiological and clinical studies suggest comorbidity between SCZ and cardiovascular-disease (CVD) risk factors, including systolic blood pressure, triglycerides, low- and high-density lipoprotein, body mass index, waist-to-hip ratio, and type 2 diabetes. Using stratified quantile-quantile plots, we show enrichment of SNPs associated with SCZ as a function of the association with several CVD risk factors and a corresponding reduction in false discovery rate (FDR). We validate this “pleiotropic enrichment” by demonstrating increased replication rate across independent SCZ substudies. Applying the stratified FDR method, we identified 25 loci associated with SCZ at a conditional FDR level of 0.01. Of these, ten loci are associated with both SCZ and CVD risk factors, mainly triglycerides and low- and high-density lipoproteins but also waist-to-hip ratio, systolic blood pressure, and body mass index. Together, these findings suggest the feasibility of using genetic-pleiotropy-informed methods for improving gene discovery in SCZ and identifying potential mechanistic relationships with various CVD risk factors.
Using reverse transcription-polymerase chain reaction and degenerate primers based on conserved sequences, a partial cDNA from Arabidopsis thaliana has been cloned that encodes part of a homologue of ...gp91-phox of NADPH oxidase. It is expressed in Arabidopsis shoots, roots and flowers and its expression in suspension cultures is up-regulated by elicitation with harpin or exposure to hydrogen peroxide.
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