Atmospheric oxidation mechanism and kinetics of 2-bromo-4,6-dinitroaniline by OH radicals - a theoretical study.

2-Bromo-4,6-dinitroaniline (BNA) is identified as a domestic-dust pollutant in urban environments, with deleterious atmospheric effects. In the present work, we studied the reaction pathways and kinetics for BNA oxidation by the OH radical using quantum-chemical methods and canonical-variational transition-state theory with small-curvature tunneling correction (CVT/SCT). OH-radial-mediated BNA oxidation was studied by considering OH addition to carbon atoms (C1 to C6) of BNA and H-atom abstraction at the -NH2 group and carbon atoms (C3 and C5) of BNA by OH radicals. It is observed that an OH-addition reaction is energetically more favorable. In addition, the rate constant was calculated for the favorable initial OH-addition reactions over the temperature range of 278 to 1000 K. The subsequent reactions for the favorable BNA-OH adduct intermediate with O2, HO2 and NO radicals are studied. We have identified the following possible end products from this BNA-oxidation reaction: (i) 2-amino-3-bromo-6-hydroperoxy-5-methyl-1-nitro-cyclohexa-2,4 dienol, (ii) 2-amino-1-bromo-6-hydroperoxy-5-methyl-3-nitro-cyclohexa-2,4-dienol, (iii) 2-amino-1-bromo-6-hydroperoxy-5-methyl-3-nitro-cyclohexa-2,4-dienol, (iv) 3-amino-4-bromo-4-hydroperoxy-8-methyl-2-nitro-6,7-dioxa-bicyclo oct-2-en-8-ol, (v) 2-amino-1-bromo-6-hydroperoxy-5-methyl-3-nitro-cyclohexa-2,4-dienol, and (vi) 3-amino-2-bromo-8-methyl-4-nitro-6,7-dioxa-bicyclo oct-3-ene-2,8-diol.

Identifying drivers of leaf water and cellulose stable isotope enrichment in Eucalyptus in northern Australia.

Several previous studies have investigated the use of the stable hydrogen and oxygen isotope compositions in plant materials as indicators of palaeoclimate. However, accurate interpretation relies on a detailed understanding of both physiological and environmental drivers of the variations in isotopic enrichments that occur in leaf water and associated organic compounds. To progress this aim we measured δ18O and δ2H values in eucalypt leaf and stem water and δ18O values in leaf cellulose, along with the isotopic compositions of water vapour, across a north-eastern Australian aridity gradient. Here we compare observed leaf water enrichment, along with previously published enrichment data from a similar north Australian transect, to Craig-Gordon-modelled predictions of leaf water isotopic enrichment. Our investigation of model parameters shows that observed 18O enrichment across the aridity gradients is dominated by the relationship between atmospheric and internal leaf water vapour pressure while 2H enrichment is driven mainly by variation in the water vapour-source water isotopic disequilibrium. During exceptionally dry and hot conditions (RH < 21%, T > 37 °C) we observed strong deviations from Craig-Gordon predicted isotope enrichments caused by partial stomatal closure. The atmospheric-leaf vapour pressure relationship is also a strong predictor of the observed leaf cellulose δ18O values across one aridity gradient. Our finding supports a wider applicability of leaf cellulose δ18O composition as a climate proxy for atmospheric humidity conditions during the leaf growing season than previously documented.

Lost therapeutic potential of monocyte-derived dendritic cells through lost tissue homing: stable restoration of gut specificity with retinoic acid.

Human monocyte-derived dendritic cells (DC) (MoDC) are utilized for immunotherapy. However, in-vitro immunological effects are often not mirrored in vivo. We studied the tissue-homing potential of MoDC. Circulating monocytes and DC expressed different tissue-homing markers and, during in-vitro development of MoDC, homing marker expression was lost resulting in a 'homeless' phenotype. Retinoic acid (RA) induced gut-homing markers (ß7 and CCR9) and a regulatory phenotype and function [decreased human leucocyte antigen D-related (HLA-DR) and increased ILT3 and fluorescein isothiocyanate (FITC-dextran uptake) in MoDC]. RA-MoDC were less stimulatory and primed conditioned T cells with a gut-homing profile (ß7(+)CLA(-)). Unlike the normal intestinal microenvironment, that from inflamed colon of ulcerative colitis (UC) patients did not induce regulatory properties in MoDC. However, RA-MoDC maintained their regulatory gut-specific properties even in the presence of UC microenvironment. Therefore, MoDC may be ineffectual for immunotherapy because they lack tissue-homing and tissue-imprinting specificity. However, MoDC rehabilitation with gut-homing potential by RA could be useful in promoting immunotherapy in pathologies such as UC.

The Integration of Proteome-Wide PTM Data with Protein Structural and Sequence Features Identifies Phosphorylations that Mediate 14-3-3 Interactions.

14-3-3s are abundant proteins that regulate essentially all aspects of cell biology, including cell cycle, motility, metabolism, and cell death. 14-3-3s work by docking to phosphorylated Ser/Thr residues on a large network of client proteins and modulating client protein function in a variety of ways. In recent years, aided by improvements in proteomics, the discovery of 14-3-3 client proteins has far outpaced our ability to understand the biological impact of individual 14-3-3 interactions. The rate-limiting step in this process is often the identification of the individual phospho-serines/threonines that mediate 14-3-3 binding, which are difficult to distinguish from other phospho-sites by sequence alone. Furthermore, trial-and-error molecular approaches to identify these phosphorylations are costly and can take months or years to identify even a single 14-3-3 docking site phosphorylation. To help overcome this challenge, we used machine learning to analyze predictive features of 14-3-3 binding sites. We found that accounting for intrinsic protein disorder and the unbiased mass spectrometry identification rate of a given phosphorylation significantly improves the identification of 14-3-3 docking site phosphorylations across the proteome. We incorporated these features, coupled with consensus sequence prediction, into a publicly available web app, called "14-3-3 site-finder". We demonstrate the strength of this approach through its ability to identify 14-3-3 binding sites that do not conform to the loose consensus sequence of 14-3-3 docking phosphorylations, which we validate with 14-3-3 client proteins, including TNK1, CHEK1, MAPK7, and others. In addition, by using this approach, we identify a phosphorylation on A-kinase anchor protein-13 (AKAP13) at Ser2467 that dominantly controls its interaction with 14-3-3.

Solid malignancies during the first year of life: A 20-year review at Red Cross War Memorial Children's Hospital, Cape Town, South Africa.

BACKGROUND: Among paediatric tumours, two groups stand out: neonatal and infantile tumours, which respectively represent 2% and 10% of paediatric tumours. The distribution of tumours in these age groups is different from that in older children. Objectives. Descriptive analysis of a cohort of patients treated for a solid malignancy at Red Cross War Memorial Children's Hospital (RCWMCH), Cape Town, South Africa. Methods. A 20-year retrospective case series review of patients aged <1 year at diagnosis was performed on data extracted from the RCWMCH oncology database. Results. Of 243 cases extracted from the database, 198 were solid tumours, of which 122 (61.1%) were included in the analysis; the 76 excluded were benign or of eye, bone or central nervous system origin and therefore did not meet the inclusion criteria. There were 38 renal malignancies (31.2%), 30 neuroblastomas (24.6%), 25 soft-tissue sarcomas (20.5%), 17 germ cell tumours/gonadal tumours (13.9%) and 12 liver tumours (9.8%). Of the patients, 119 (97.5%) had surgery, 91 (74.6%) had chemotherapy and 10 (8.2%) had radiotherapy. Tumour group 5-year survival was 78.5% for neuroblastic tumours, 79.0% for nephroblastomas, 81.5% for hepatoblastomas, 62.5% and 54.2% for rhabdomyosarcoma and non-rhabdomyosarcoma soft-tissue sarcomas, respectively, and 79.5% for malignant extracranial and extragonadal germ cell tumours. For the entire cohort, the mean follow-up was 46 months, with an estimated 5-year overall survival of 74.6%. Mortality was 21.5% and loss to follow-up 6.6%. Conclusion. The distribution of tumours differs slightly from the literature, with a predominance of renal tumours over neuroblastomas. The overall mortality rate of 21.5%, the surgical complication rate of 10.9% and the 5-year overall survival of 74.6% correspond with the literature, supporting the view that a paediatric hospital in a middle-income country can achieve results similar to those in higher-income countries when international protocols are applied by a dedicated multidisciplinary team.

Age-growth relationships, temperature sensitivity and palaeoclimate-archive potential of the threatened Altiplano cactus Echinopsis atacamensis.

The tall (>4 m), charismatic and threatened columnar cacti, pasacana [Echinopsis atacamensis (Vaupel) Friedrich & G.D. Rowley)], grows on the Bolivian Altiplano and provides environmental and economic value to these extremely cold, arid and high-elevation (~4000 m) ecosystems. Yet very little is known about their growth rates, ages, demography and climate sensitivity. Using radiocarbon in spine dating time series, we quantitatively estimate the growth rate (5.8 and 8.3 cm yr-1) and age of these cacti (up to 430 years). These data and our field measurements yield a survivorship curve that suggests precipitation on the Altiplano is important for this species' recruitment. Our results also reveal a relationship between nighttime temperatures on the Altiplano and the variation in oxygen isotope values in spines (δ18O). The annual δ18O minimums from 58 years of in-series spine tissue from pasacana on the Altiplano provides at least decadal proxy records of temperature (r = 0.58; P < 0.0001), and evidence suggests that there are longer records connecting modern Altiplano temperatures to sea-surface temperatures (SSTs) in the Atlantic Ocean. While the role of Atlantic SSTs on the South American Summer Monsoon (SASM) and precipitation on the Bolivian Altiplano is well described, the impact of SSTs on Altiplano temperatures is disputed. Understanding the modern impact of SSTs on temperature on the Altiplano is important to both understand the impact of future climate change on pasacana cactus and to understand past climate changes on the Altiplano. This is the best quantitative evidence to date of one of the oldest known cactus in the world, although there are likely many older cacti on the Altiplano, or elsewhere, that have not been sampled yet. Together with growth, isotope and age data, this information should lead to better management and conservation outcomes for this threatened species and the Altiplano ecosystem.

Variant cell lines selected for alterations in the function of the hyaluronan receptor CD44 show differences in glycosylation.

CD44 is a major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan (HA). However, the ability of CD44 to bind ligand is strictly regulated. Three activation states of CD44 have been demonstrated: (a) inactive; (b) inducible (by certain CD44-specific mAb); and (c) constitutively active. Starting with two parental cell lines expressing CD44 in the inactive state, a pre-B cell (RAW 253) and a fibroblast (L cells), we used fluorescence-activated cell sorting with fluorescein-conjugated hyaluronan in the presence of inducing mAb to derive variant cell lines with CD44 in the inducible state. Constitutively active derivatives were isolated from the inducible variants by a further round of fluorescence-activated cell sorting in the absence of inducing antibody. However, constitutively active variants could not be isolated directly from parental cells expressing CD44 in the inactive state. These results suggest that two genetic events must occur to obtain an active CD44-HA receptor from an inactive receptor. Variant and parental cell-derived CD44 molecules exhibited differences in migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were partly attributable to differences in N-linked glycosylation. Furthermore, culture in tunicamycin for 2-3 d converted parental and inducible cell lines into cells showing constitutive CD44-mediated HA binding. Also, removal of cell surface glycosaminoglycan chains by culture of cells in p-nitrophenyl beta-D-xylopyranoside or treatment with chondroitinase ABC resulted in conversion of cells with an inactive CD44 receptor to an inducible state. These results indicate that carbohydrate side chains of CD44 and/or other molecules on the cell surface that interact with CD44 are potentially involved in regulating the HA-binding function of CD44 on the cell surface.

A novel population of human CD56+ human leucocyte antigen D-related (HLA-DR+) colonic lamina propria cells is associated with inflammation in ulcerative colitis.

Ulcerative colitis (UC) involves inappropriate mucosal immune responses to intestinal microbiota. Gut dendritic cells (DC) are central immunoregulators of the response to commensal bacteria, and the subset of CD11c(+) cells within the human leucocyte antigen D-related (HLA-DR(+)) lineage (lin)(-/dim) population are activated in inflammatory bowel disease. We hypothesized that CD11c(-) cells within this population may also be involved in intestinal inflammation. HLA-DR(+) lin(-/dim) cells were identified in freshly isolated lamina propria mononuclear cells by multi-colour flow cytometry in 54 UC patients and 22 controls. Proportion and number of CD11c(+) and CD11c(-) cells, and surface expression of activation markers CD40, CD86, Toll-like receptor (TLR)-2, TLR-4, and CD56(+)[natural killer (NK) marker], were determined. Cytokine production was assessed by intracellular staining. Lamina propria colonic CD11c(-) HLA-DR(+) lin(-/dim) cells were increased significantly in inflamed and 'non-inflamed' UC tissue, compared with control tissue. CD11c(+) HLA-DR(+) lin(-/dim) cells were unchanged. Fewer CD11c(-) cells expressed activation markers and produced intracellular cytokines than their CD11c(+) counterparts, and they were weakly stimulatory in mixed leucocyte reactions. Few CD11c(-) cells expressed blood plasmacytoid DC markers, but a major subset expressed high levels of CD56. CD11c(-) cells decreased after inflammation resolved. Intestinal inflammation in UC is associated with the presence of cells that share phenotypic features of both DC and NK cells. This novel population of human colonic CD56(+) HLA-DR(+) cells may play a role in immune regulation or tissue repair. Their increase in quiescent UC may be a marker of subclinical inflammation.

Site-specific de-N-glycosylation of CD44 can activate hyaluronan binding, and CD44 activation states show distinct threshold densities for hyaluronan binding.

CD44 is a cell surface receptor for the glycosaminoglycan hyaluronan (HA). Not all CD44-positive cells bind HA, and binding ability is strictly regulated. Three different HA binding states have been defined: inactive, inducible (by certain CD44-specific monoclonal antibodies), and constitutively active. The observation that sets of genetically related cell lines representing different HA binding states showed correlated differences in N-glycosylation of CD44, and that inhibition of N-glycosylation enhanced HA binding (Lesley et al., J. Exp. Med., 182: 431-437, 1995) led us to examine directly whether specific N-glycosylation site modifications were involved in regulating the HA binding function. CD44-negative, -active, and inducible cell lines were stably transfected with mutant constructs in which each of the five N-glycosylation sites of murine CD44 had been separately inactivated. Ability to bind soluble HA was examined over a range of CD44 expression levels. For the active cell line, AKR1, transfectants for all N-glycosylation mutants bound HA as well as did transfectants for wild type CD44. No inhibitory effects of inactivating specific N-glycosylation sites were observed. HA binding was activated when two of the mutant constructs were transfected into a novel CD44-negative inducible cell line. Inactivation of N-glycosylation sites at residues 25 or 120 converted the inducible cell line to constitutively active, whereas inactivation of other sites had little or no effect. Fusion proteins secreted from inactive, inducible, or active cell lines were purified, bound to beads, and assayed for HA binding activity by flow cytometric analysis. Fusion proteins derived from inactive, inducible, and constitutively active cells exhibited three distinguishable "threshold" densities required for HA binding ability. The results imply that the CD44 molecules produced in cells in these three activation states have intrinsic differences in HA binding function. Treatment of the fusion proteins with neuraminidase altered the HA binding state, and glycosylation mutations that affected the phenotype of the inducible cell line lowered the threshold required for HA binding of CD44-immunoglobulin fusion proteins derived from the inducible cell line. Thus, alterations of glycosylation of CD44 itself can affect HA binding ability as manifested by a change in HA binding state.

Variable alterations of the microbiota, without metabolic or immunological change, following faecal microbiota transplantation in patients with chronic pouchitis.

Faecal microbiota transplantation (FMT) is effective in the treatment of Clostridium difficile infection, where efficacy correlates with changes in microbiota diversity and composition. The effects of FMT on recipient microbiota in inflammatory bowel diseases (IBD) remain unclear. We assessed the effects of FMT on microbiota composition and function, mucosal immune response, and clinical outcome in patients with chronic pouchitis. Eight patients with chronic pouchitis (current PDAI ≥7) were treated with FMT via nasogastric administration. Clinical activity was assessed before and four weeks following FMT. Faecal coliform antibiotic sensitivities were analysed, and changes in pouch faecal and mucosal microbiota assessed by 16S rRNA gene pyrosequencing and (1)H NMR spectroscopy. Lamina propria dendritic cell phenotype and cytokine profiles were assessed by flow cytometric analysis and multiplex assay. Following FMT, there were variable shifts in faecal and mucosal microbiota composition and, in some patients, changes in proportional abundance of species suggestive of a "healthier" pouch microbiota. However, there were no significant FMT-induced metabolic or immunological changes, or beneficial clinical response. Given the lack of clinical response following FMT via a single nasogastric administration our results suggest that FMT/bacteriotherapy for pouchitis patients requires further optimisation.

Subpopulations of peripheral blood dendritic cells show differential susceptibility to infection with a lymphotropic strain of HIV-1.

Blood dendritic cells (DC) express CD4 and are susceptible to HIV infection. By electron microscopy two morphologically distinct types of DC were identified in peripheral blood. Only one of these two types was susceptible to infection with a lymphotropic strain of HIV-1. By FACS two populations could be defined based on the expression of CD11c. The morphology of cultured FACS-purified CD11c negative DC was similar to that DC population shown to be susceptible to infection with the lymphotropic strain of HIV. Furthermore after several hours in culture CXCR4, the co-receptor for lymphotropic strains of HIV-1, was expressed at a significantly higher level on the CD11c negative DC than on the CD11c positive cells. This study suggests that there are subpopulations of DC that show differences in susceptibility to infection with some strains of HIV-1.

Relationship between V3 sequences of HIV type 1 from plasma, T cells, and dendritic cells suggests that plasma virus may arise from different cell sources.

Nucleotide sequences of HIV-1 from plasma virus RNA and proviral DNA extracted from blood dendritic cells (DCs) and from T cells were analyzed to determine whether blood DCs may harbor a restricted population of virus variants. The sequence of the V3 loop and 51 bases from the 3' flanking region were determined in four patients not receiving antiviral therapy. There was no evidence of a unique or more restricted population of variants in DCs for any of the four patients studied. However, for one patient there was evidence of differences between plasma virus and virus in the T cell population, with virus in the plasma showing a closer relationship to DC-derived sequences.

Antioxidant-mediated attenuation of the induction of cytochrome P450BM-3(CYP102) by ibuprofen in Bacillus megaterium ATCC 14581.

Bacillus megaterium contains a soluble cytochrome P450 termed BM-3, which is highly inducible by barbiturates, peroxisome proliferators, and nonsteroidal antiinflammatory drugs. In rats and mice, the chronic administration of peroxisome proliferators induces a sustained oxidative stress in hepatic tissue and may be responsible for the nongenotoxic carcinogenesis observed with prolonged treatment. Here it is shown that ibuprofen induces a variety of enzymes associated with the oxidative stress response in Bacillus, including catalase, glucose-6-phosphate-dehydrogenase, and aldehyde reductase in a dose-related manner. Furthermore, evidence is presented to show that the expression of cytochrome P450 in Bacillus is associated with a marked depletion in cellular glutathione levels and that it renders these cells considerably more sensitive to oxidant insult. Finally, this work reports that a variety of structurally diverse antioxidants such as ascorbic acid, reduced glutathione, alpha-tocopherol acetate and the artificial antioxidant, butylated hydroxyanisole, all dramatically attenuate the expression of the cytochrome P450BM-3 gene and its repressor, Bm3R1, following ibuprofen treatment. These observations provide the first evidence that the expression of cytochrome P450 genes can lead to increased oxidant sensitivity but can be strongly modulated by dietary and artificial antioxidants, as well as antioxidant enzymes. The important implications of this phenomenon are also discussed.

Measurement of L-carnitine and acylcarnitines in body fluids and tissues in children and in adults.

We have developed a reliable and validated radio-enzymatic method for the assay of L-carnitine and acylcarnitines, using a modification of existing methods. The sensitivity of the assay is 10 mumol/l using 10 microliters of plasma or urine. It is also suitable for measurements of carnitine in a 10 mg sample of liver or muscle obtained by percutaneous biopsy. The use of N-ethylmaleimide in the reaction mixture together with an excess of [1-14C]acetyl CoA ensures that the reaction proceeds to completion and a linear response is obtained. Using this method control ranges have been established for plasma and urine carnitine concentrations in healthy children and adults, and for the carnitine content of liver and muscle in adults. No significant difference was found between fasting and post-prandial plasma carnitine levels. An age-related increase was found in urinary total carnitine and acylcarnitine concentration throughout childhood. These data provide a reliable basis for studies of patients with abnormal carnitine and acylcarnitine metabolism, distribution and excretion.

Critical caring. Promoting good end-of-life care in the intensive care unit.

Changing the culture in the ICU to include palliative care interventions along with curative interventions is already underway. Further work is needed, however. This is a role for the critical care nurse. Critical care nurses can be involved in research and education to enhance their future practice in end-of-life care. Research to establish evidence-based protocols for use in patients who require palliative care in the ICU needs to be done. Critical care nurses can prepare themselves for carrying or dying patients by attending palliative care seminars and continuing education courses or by taking a short clinical sabbatical or internship in a local hospice to observe and help give end-of-life care. Hospice nurses can be invited to the ICU to give inservice sessions and to help nurses and other staff understand the transition to dying, including the services that need to be offered to the patient and the family. Nurses from the hospital palliative care team can consult and be available for follow-up. Promoting good end-of-life care should be a goal for all intensive care nurses and critical care units. This goal is reached one patient at a time.

A criterion-referenced measure of latex allergy knowledge.

The exponential use of latex products has increased patients' and health care workers' sensitization to natural rubber latex. The purpose of this research study was to develop and test a set of items designed to measure professional nurses' knowledge about natural rubber latex reactions and interventions appropriate for latex precautions. The items were developed from research reports in the literature and changed to reflect the suggestions offered by experienced perioperative nurses (pilot = 8) and three nationally known experts in latex allergy responses. The revised form was mailed to the home or distributed at the workplace of perioperative nurses (n = 158). Criteria for item retention were < .90 difficulty index and clinical relevance. Certified nurses (i.e., CNORs) scored significantly higher than other nurses who took the test at the same time (t-test = 2.00, P = .05). A pretest and posttest evaluation of the revised 20 items was conducted before and after a latex allergy continuing education program during the spring of 1998. The current knowledge test (i.e., version 1.1) has reliability (alpha = .80); content validity (CVI = .92); and accumulated evidence of construct validity (preinstruction mean = 14.16, postinstruction mean = 17.15, t-paired = 10.54, df = 70, P = < .001). An empirically developed professional nurse latex allergy competency test suitable for clinical use was the outcome of this research study.

A mechanistic role for leptin in human dendritic cell migration: differences between ileum and colon in health and Crohn's disease.

Dendritic cells (DC) migrate to lymph nodes on expression of C-C motif chemokine receptor 7 (CCR7) and control immune activity. Leptin, an immunomodulatory adipokine, functions via leptin receptors, signaling via the long isoform of receptor, LepRb. Leptin promotes DC maturation and increases CCR7 expression on blood DC. Increased mesenteric fat and leptin occur early in Crohn's disease (CD), suggesting leptin-mediated change in intestinal CCR7 expression on DC as a pro-inflammatory mechanism. We have demonstrated CCR7 expression and capacity to migrate to its ligand macrophage inflammatory protein 3ß in normal human ileal DC but not colonic or blood DC. In CD, functional CCR7 was expressed on DC from all sites. Only DC populations containing CCR7-expressing cells produced LepRb; in vitro exposure to leptin also increased expression of functional CCR7 in intestinal DC in a dose-dependent manner. In conclusion, leptin may regulate DC migration from gut, in homeostatic and inflammatory conditions, providing a link between mesenteric obesity and inflammation.