Biomedical subjects
A F Taylor
Publications and source records attributed to A F Taylor.
Large-scale forest girdling shows that current photosynthesis drives soil respiration.
The respiratory activities of plant roots, of their mycorrhizal fungi and of the free-living microbial heterotrophs (decomposers) in soils are significant components of the global carbon balance, but their relative contributions remain uncertain. To separate mycorrhizal root respiration from heterotrophic respiration in aboreal pine forest, we conducted a large-scale tree-girdling experiment, comprising 9 plots each containing about 120 trees. Tree-girdling involves stripping the stem bark to the depth of the current xylem at breast height terminating the supply of current photosynthates to roots and their mycorrhizal fungi without physically disturbing the delicate root-microbe-soil system. Here we report that girdling reduced soil respiration within 1-2 months by about 54% relative to respiration on ungirdled control plots, and that decreases of up to 37% were detected within 5 days. These values clearly show that the flux of current assimilates to roots is a key driver of soil respiration; they are conservative estimates of root respiration, however, because girdling increased the use of starch reserves in the roots. Our results indicate that models of soil respiration should incorporate measures of photosynthesis and of seasonal patterns of photosynthate allocation to roots.
Treatment of childhood asthma with anti-immunoglobulin E antibody (omalizumab).
BACKGROUND AND OBJECTIVE: There seems to be a strong causal relationship between allergy and the origins of asthma. Susceptibility to both is determined by a combination of genetics and environment acting through a complex network of cytokines. Nearly 90% of affected children have positive skin tests indicating the presence of specific immunoglobulin E (IgE), with sensitivity to house dust mite, Alternaria, cockroach, cat, and dog most closely linked to the disease. Greater exposure to house dust mite during infancy leads to earlier onset of wheezing, and elevated serum IgE levels correlate with the appearance of asthma symptoms. Specific IgE binds to high-affinity (FcepsilonRI) receptors on mast cells and basophils. The IgE-mediated reactions that follow exposure of sensitized mast cells to an allergen are designated early- and late-phase asthmatic responses (EAR and LAR). EAR is characterized by release of histamine and other preformed mediators within 1 hour of allergen exposure. It is often followed by LAR, an infiltration of the airways by inflammatory cells associated with an episode of more prolonged, and usually more severe airflow obstruction, 4 to 8 hours after antigen exposure. Chronic airway symptoms result from persistent LAR caused by continuous allergen exposure. IgE antibodies are capable of passive transfer of both EAR and LAR sensitivity. IgE-mediated mast cell activation contributes to chronic tissue eosinophilia and airway remodeling, with permanent loss in pulmonary function. Omalizumab (rhuMAb-E25) is a recombinant, humanized, monoclonal anti-IgE antibody of mouse origin developed for the treatment of IgE-mediated diseases. Omalizumab binds to free IgE at the same site as the high-affinity receptor. Although it attaches to free IgE, it does not bind to IgA, IgG, or cell-bound IgE. It therefore does not induce cross-linking of cell-bound IgE, which would lead to the release of allergic mediators. It has been reported to decrease serum IgE levels in a dose-dependent manner, inhibit EAR and LAR, and cause a down-regulation of FcepsilonRI receptors on basophils. Omalizumab has been reported to be safe and effective in improving asthma control and reducing the requirement for oral and inhaled corticosteroids. This double-blind, randomized, placebo-controlled study evaluated the safety, steroid-sparing effects, and impact on disease exacerbations of omalizumab in the treatment of childhood asthma. Methods. Participants were 334 males and premenarchal females aged 6 to 12 years, with moderate to severe allergic asthma requiring treatment with inhaled corticosteroids. During a run-in phase, all children were switched to equivalent doses of beclomethasone dipropionate (BDP), and the dose was adjusted to assure maintenance of asthma control achieved with previous corticosteroid treatment. Children were randomized to subcutaneously administered placebo (N = 109) or omalizumab (N = 225) at a dose based on body weight and initial serum IgE (0.016 mg/kg/IgE [IU/mL] per 4 weeks). BDP dose (initial range 168-420 microg/d) was kept stable for 16 weeks (stable-steroid phase), reduced over 8 weeks to the minimum effective dose (steroid-reduction phase), and maintained constant for the final 4 weeks. RESULTS: More participants in the omalizumab group decreased their BDP dose, and their reduction was greater than that of the placebo group (median reduction 100% vs 66.7%). BDP was withdrawn completely in 55% of the omalizumab group versus 39% of the placebo group. The incidence and the frequency of asthma exacerbations requiring treatment with doubling of BDP dose or systemic corticosteroids were lower in the omalizumab group. The treatment differences were statistically significant during the steroid-reduction phase, during which fewer participants in the omalizumab group had asthma exacerbation episodes (18.2% vs 38.5%), and the mean number of episodes per patient was smaller than with placebo (0.42 vs 2.72). Five asthma exacerbations requiring hospitalization all occurred in the placebo group. Participants' and investigators' global evaluations of treatment effectiveness were more favorable for omalizumab than placebo. Investigators rated effectiveness excellent for 31.5% of the omalizumab group versus 16.3% of the placebo group and good for 44.7% of the omalizumab group versus 32.7% of the placebo group. There was little change in asthma symptom scores or spirometry measurements during either the stable-steroid or steroid dose-reduction phase, with minimal differences between the treatment groups. The requirement for rescue medication in the omalizumab group during both the stable-steroid and steroid dose-reduction phases was consistently lower than at baseline. At week 28, the median number of puffs of rescue medication taken daily was 0 in the omalizumab group and 0.46 in the placebo group. The change from baseline was significant in favor of omalizumab. (ABSTRACT TRUNCATED)
Glutamate signalling in bone.
The identification of novel signalling pathways in a tissue provides new avenues for pharmacological manipulation of tissue function. Where the pathway concerned is one that has been the subject of extensive research in another body system, progress towards new therapies can be rapid. The discovery that glutamate has functions in bone that share striking similarities with its role in synaptic neurotransmission opens the way to manipulate skeletal pathophysiology using modulators of glutamate release, uptake or receptor function. The purpose of this review is to describe the way that a role for glutamate as a signalling molecule in bone was discovered, to summarise the evidence for this role. In addition, it will identify points that are unresolved, to highlight areas where new research could provide significant advances. Furthermore, it will indicate how studies already performed but analysed without consideration of the non-neuronal functions of modulators of glutamate signalling, could contain information of significant value for the advance of therapeutic approaches to bone diseases.
The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair.
The RecBCD enzyme is required for homologous recombination and DNA repair in Escherichia coli. The structure and function of RecBCD enzyme is altered on its interaction with the recombination hotspot Chi (5'-GCTGGTGG-3'). It has been hypothesized that the RecD subunit plays a role in Chi-dependent regulation of enzyme activity [Thaler, D. S., Sampson, E., Siddiqi, I., Rosenberg, S. M., Stahl, F. W. & Stahl, M. (1988) in Mechanisms and Consequences of DNA Damage Processing, eds. Friedberg, E. & Hanawalt, P. (Liss, New York), pp. 413-422; Churchill, J. J., Anderson, D. G. & Kowalczykowski, S. C. (1999) Genes Dev. 13, 901-911]. We tested the hypothesis that the RecD subunit inhibits recombination by deleting recD from the nuclease- and recombination-deficient mutant recB(D1080A)CD. We report here that the resulting strain, recB(D1080A)C, was proficient for recombination and DNA repair. Recombination proficiency was accompanied by a change in enzyme activity: RecB(D1080A)C enzyme loaded RecA protein onto DNA during DNA unwinding whereas RecB(D1080A)CD enzyme did not. Together, these genetic and biochemical results demonstrate that RecA loading by RecBCD enzyme is required for recombination in E. coli cells and suggest that RecD interferes with the enzyme domain required for its loading. A nuclease-dependent signal appears to be required for a change in RecD that allows RecA loading. Because RecA loading is not observed with wild-type RecBCD enzyme until it acts at a Chi site, our observations support the view that RecD inhibits recombination until the enzyme acts at Chi.
Diversity and abundance of resupinate thelephoroid fungi as ectomycorrhizal symbionts in Swedish boreal forests.
Resupinate thelephoroid fungi (hereafter called tomentelloid fungi) have a world-wide distribution and comprise approximately 70 basidiomycete species with inconspicuous, resupinate sporocarps. It is only recently that their ability to form ectomycorrhizas (EM) has been realized, so their distribution, abundance and significance as mycobionts in forest ecosystems is still largely unexplored. In order to provide baseline data for future ecological studies of tomentelloid fungi, we explored their presence and abundance in nine Swedish boreal forests in which the EM communities had been analysed. Phylogenetic analyses were used to compare the internal transcribed spacer of nuclear ribosomal DNA (ITS rDNA) sequence data obtained from mycobionts on single ectomycorrhizal tips with that obtained from sporocarps of identified tomentelloid fungi. Five species of Tomentella and one species of Pseudotomentella were identified as ectomycorrhizal fungi. The symbiotic nature of Tomentella bryophila, T. stuposa, T. badia and T. atramentaria is demonstrated for the first time. T. stuposa and Pseudotomentella tristis were the most commonly encountered tomentelloid fungi, with the other species, including T. sublilacina, only being recorded from single stands. Overall, tomentelloid fungi were found in five of the studies, colonizing between 1 and 8% of the mycorrhizal root tips. Two of the five sites supported several tomentelloid species. Tomentelloid fungi appear to be relatively common ectomycorrhizal symbionts with a wide distribution in Swedish coniferous forests. The results are in accordance with accumulating data that fungal species which lack conspicuous sporocarps may be of considerable importance in EM communities.
Natural (13)C abundance reveals trophic status of fungi and host-origin of carbon in mycorrhizal fungi in mixed forests.
Fungi play crucial roles in the biogeochemistry of terrestrial ecosystems, most notably as saprophytes decomposing organic matter and as mycorrhizal fungi enhancing plant nutrient uptake. However, a recurrent problem in fungal ecology is to establish the trophic status of species in the field. Our interpretations and conclusions are too often based on extrapolations from laboratory microcosm experiments or on anecdotal field evidence. Here, we used natural variations in stable carbon isotope ratios (delta(13)C) as an approach to distinguish between fungal decomposers and symbiotic mycorrhizal fungal species in the rich sporocarp flora (our sample contains 135 species) of temperate forests. We also demonstrated that host-specific mycorrhizal fungi that receive C from overstorey or understorey tree species differ in their delta(13)C. The many promiscuous mycorrhizal fungi, associated with and connecting several tree hosts, were calculated to receive 57-100% of their C from overstorey trees. Thus, overstorey trees also support, partly or wholly, the nutrient-absorbing mycelia of their alleged competitors, the understorey trees.
Regulation of homologous recombination: Chi inactivates RecBCD enzyme by disassembly of the three subunits.
We report here an unusual mechanism for enzyme regulation: the disassembly of all three subunits of RecBCD enzyme after its interaction with a Chi recombination hot spot. The enzyme, which is essential for the major pathway of recombination in Escherichia coli, acts on linear double-stranded DNA bearing a Chi site to produce single-stranded DNA substrates for strand exchange by RecA protein. We show that after reaction with DNA bearing Chi sites, RecBCD enzyme is inactivated and the three subunits migrate as separate species during glycerol gradient ultracentrifugation or native gel electrophoresis. This Chi-mediated inactivation and disassembly of purified RecBCD enzyme can account for the previously reported Chi-dependent loss of Chi activity in E. coli cells containing broken DNA. Our results support a model of recombination in which Chi regulates one RecBCD enzyme molecule to make a single recombinational exchange ('one enzyme-one exchange' hypothesis).
A stimulatory RNA associated with RecBCD enzyme.
RecBCD enzyme acts in the major pathway of homologous recombination of linear DNA in Escherichia coli. The enzyme unwinds DNA and is an ATP-dependent double-strand and single-strand exonuclease and a single-strand endonuclease; it acts at Chi recombination hotspots (5'-GCTGGTGG-3') to produce a recombinogenic single-stranded DNA 3'-end. We found that a small RNA with a unique sequence of approximately 24 nt was tightly bound to RecBCD enzyme and co-purified with it. When added to native enzyme this RNA, but not four others, increased DNA unwinding and Chi nicking activities of the enzyme. In seven similarly active enzyme preparations the molar ratio of RNA molecules to RecBCD enzyme molecules ranged from 0.2 to <0.008. These results suggest that, although this unique RNA is not an essential enzyme subunit, it has a biological role in stimulating RecBCD enzyme activity.
Genomics, Chi sites and codons: 'islands of preferred DNA pairing' are oceans of ORFs.
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Surgical Pearl: The use of multiple different tissue specimens on the same glass slide to enhance the efficiency of frozen section preparation in Mohs micrographic surgery.
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Monomeric RecBCD enzyme binds and unwinds DNA.
RecBCD enzyme is a multifunctional nuclease that is essential for the major pathway of homologous genetic recombination in Escherichia coli. It has a potent helicase activity that uses ATP hydrolysis to unwind very long stretches of DNA. The functional form of RecBCD enzyme has been unclear, since M(r) of 250,000-655,000 have been previously reported. We have isolated two oligomeric forms of the enzyme, one (monomeric) containing a single copy of the RecB, RecC, and RecD polypeptides, and the other (dimeric) containing two copies of each polypeptide. We show here that the monomeric form of the enzyme (M(r) approximately 330,000) can form a stable initiation complex on the end of ds DNA. Depending on the nature of the ds end, KD estimates ranged from approximately 0.1 nM to approximately 0.7 nM in the presence of Mg2+ ions, which enhanced but was not required for binding. We further showed that the complex of monomeric RecBCD enzyme and a ds DNA end was competent to unwind DNA. A general model for the action of helicases has been proposed that uses repeated conformational changes between two states of a complex between DNA and a dimeric form of the enzyme. Our results make such a model unlikely for RecBCD enzyme.
Strand specificity of nicking of DNA at Chi sites by RecBCD enzyme. Modulation by ATP and magnesium levels.
RecBCD enzyme is essential for the major pathway of homologous recombination of linear DNA in Escherichia coli. It is a potent nuclease and helicase and, during its unwinding of double-stranded DNA, makes single-strand scissions in the vicinity of Chi recombination hot spots. We report here that both the strand that is cut and the position of the cuts relative to Chi depended on the ATP to Mg2+ ratio. With ATP in excess, Chi-dependent nicks occurred, as we have previously reported, four to six nucleotides to the 3'-side of the Chi octamer (5'-GCTGGTGG-3') and were detected only on the strand bearing that sequence. Three differences were seen with Mg2+ in excess. 1) Chi-dependent 3'-ends were produced on the GCTGGTGG-containing strand closer to and within the Chi octamer. 2) Chi-dependent cuts occurred on the complementary DNA strand. 3) RecBCD enzyme destroyed the 3'-terminated strand of DNA from its entry point up to the vicinity of the Chi site, as others have previously reported. We show here that, with Mg2+ in excess, the enzyme continued to travel along DNA, after encountering a Chi site, releasing both strands of the DNA distal to Chi as single strands. We discuss potential biological consequences of these two modes of RecBCD enzyme-Chi interaction.
The initiation and control of homologous recombination in Escherichia coli.
The chromosome of Escherichia coli recombines at low frequency when it is an intact circle but recombines at high frequency when it is broken, for example by X-rays, or when a linear DNA fragment is introduced into the cell during conjugation or transduction. The high recombinogenicity of double-strand (ds) DNA ends is attributable to RecBCD enzyme, which acts on ds DNA ends and is essential for recombination and ds DNA break repair. RecBCD enzyme initiates DNA unwinding at ds DNA ends, and its nuclease activity is controlled by Chi sites (5' G-C-T-G-G-T-G-G 3') in such a way that the enzyme produces a potent single-stranded DNA substrate for homologous pairing by RecA and single-stranded DNA binding proteins. We discuss a unifying model for recombination and ds DNA break repair, based upon the enzymic activities of these and other proteins and upon the behaviour of E. coli mutants altered in these proteins.
Effect of ductal patency on organ blood flow and pulmonary function in the preterm baboon with hyaline membrane disease.
OBJECTIVE: To examine the effect of early ductal ligation vs. maintenance of ductal patency on vital organ perfusion and pulmonary function in premature baboons with hyaline membrane disease. DESIGN: Randomized, controlled interventional study to compare early ligation with formalin infiltration of the ductus arteriosus. SETTING: Animal care facility at a dedicated research foundation. SUBJECTS: Eighteen premature baboons delivered by hysterotomy at 140 +/- 2 day gestation. INTERVENTION: Nine premature baboons underwent formalin infiltration of the ductus arteriosus (group 1), and nine underwent ductal ligation (group 2). Surgical ligation or formalin infiltration was performed at 2 to 4 hrs of age. Animals were maintained on mechanical ventilation and ventilator parameters were adjusted to maintain PaO2 and PaCO2 within the physiologic range. MAIN OUTCOME MEASURES: Left ventricular output indexed to body weight and vital organ perfusion were measured at 24 hrs of age by the radiolabeled microsphere method. Lung mechanics, including lung wet/dry weight ratio, total lung water, static compliance and functional residual capacity were measured immediately following euthanasia. RESULTS: Total pulmonary blood flow was significantly lower (p = .0001) in group 2 (mean = 94 mL/min/kg), compared with group 1 (mean = 287 mL/min/kg). Systemic blood flow and effective pulmonary blood flow were higher in group 1 (p = .07). No significant difference between groups in absolute organ blood flow was noted, although flow as a percent of left ventricular index was significantly higher in all organs except the kidney in group 2. There was no difference in arterial blood gas values, parameters of mechanical ventilation, percent lung water, or postmortem measurement of lung mechanics between groups. CONCLUSION: Early ductal ligation did not result in improved cardiac output, increased organ blood flow, or improved pulmonary function. We postulate that gradual constriction of the ductus arteriosus may play an important role in successful cardiovascular adaptation in the premature infant. While it is clear that premature infants with symptomatic patent ductus arteriosus often benefit from ductal closure, we question the practice of prophylactic early ductal closure.
Pemphigus foliaceus and pemphigus vulgaris autoantibodies react with the extracellular domain of desmoglein-1.
Pemphigus foliaceus is associated with an autoimmune response against desmoglein-1; however, the fine specificity of these autoantibodies and the role that they play in pathogenesis have not yet been elucidated. In an attempt to develop a system to facilitate the detection and characterization of this antigen/antibody system, recombinant human desmoglein-1 was expressed in COS-1 cells, a mammalian epithelial cell line. The desmoglein-1 transgene product was shown to be expressed on the surface of the COS-1 cells in the appropriate transmembrane orientation. All pemphigus foliaceus sera (endemic form, n = 24; nonendemic form, n = 7) reacted strongly with nonpermeabilized desmoglein-1-transfected cells, exhibiting a punctate cell-surface staining pattern. This reactivity against the desmoglein-1 ectodomain was predominantly an IgG4-restricted response and was calcium dependent. Ten of 18 pemphigus vulgaris sera also reacted with the extra-cellular domain of recombinant desmoglein-1. Use of this eukaryotic expression system should greatly facilitate further characterization of the anti-desmoglein-1 autoimmune response associated with pemphigus foliaceus and pemphigus vulgaris and may aid in determining its pathogenic relevance.
Development of an ELISA to detect anti-BP180 autoantibodies in bullous pemphigoid and herpes gestationis.
Autoantibodies associated with the subepidermal blistering disorders bullous pemphigoid and herpes gestationis react with a 180-kD transmembrane hemidesmosomal protein, designated BP180. The BP180 ectodomain is composed of a series of interrupted collagen triple helical domains. Located on one of the noncollagenous extracellular segments of this protein is an immunodominant epitope, designated MCW-1, recognized by patient autoantibodies. In this investigation we have developed an enzyme-linked immunosorbent assay system to detect antibody reactivity against the MCW-1 epitope with the use of a bacterial fusion protein containing the BP180 autoantibody-reactive site. The following sera were assayed for reactivity with this recombinant protein: bullous pemphigoid (n = 62), herpes gestationis (n = 28), endemic pemphigus foliaceus (n = 17), lupus erythematosus (n = 15), and normal human sera (n = 22). This enzyme-linked immunosorbent assay-based protocol was shown to be highly specific (98.3%) in detecting autoantibody activity in bullous pemphigoid and herpes gestationis patients. Fifty-three percent of bullous pemphigoid sera and 71% of herpes gestations sera, but none of the control sera, yielded positive results in this assay. Of the patient sera that were known to react with full-length BP180, almost all showed reactivity with the MCW-1 antigenic site of this protein. Autoantibodies detected in this assay were predominantly of the immunoglobulin G class. The results presented here lend support to the hypothesis that this well-defined antigen/antibody system may be relevant in pathogenesis.
Hormonal response of the premature primate to operative stress.
There are few data on the hormonal response to operation in the premature infant. Studies examining the response of newborn human infants have been performed on patients beyond the first few days of life, where some adaptation to postnatal life has occurred. This study evaluated the response of the newly born premature primate to surgical stress. Premature baboons (75% gestation) were intubated, mechanically ventilated and underwent thoracotomy at 2 hours of life with exposure of the ductus arteriosus (PDA). In group 1, formalin was infiltrated to keep the ductus patent. In group 2, the PDA was ligated. Controls had no operation. Blood was drawn at 0, 6, 24, 48, 72, and 96 hours of age. Echocardiograms were performed to confirm patency or closure of the ductus and to monitor cardiac function. Epinephrine, norepinephrine, renin, and cortisol levels were measured. Cortisol levels rose in all groups. Operation stimulated a marked increase in catecholamine and renin levels in both operative groups, which was more marked in the group with PDA ligation at 24 hours. These data reflect expected pathophysiology since early PDA ligation exerts additional hemodynamic demand on the heart. In conclusion, the premature primate is able to mount a significant and severity-dependent endocrine response to stress.