The role of absC,a novel regulatory gene for secondary metabolism,in zinc‐dependent antibiotic production in Streptomyces coelicolor A3(2)

The availability of zinc was shown to have a marked influence on the biosynthesis of actinorhodin in Streptomyces coelicolor A3(2). Production of actinorhodin and undecylprodigiosin was abolished when a novel pleiotropic regulatory gene, absC, was deleted, but only when zinc concentrations were low. AbsC was shown to control expression of the gene cluster encoding production of coelibactin, an uncharacterized non‐ribosomally synthesized peptide with predicted siderophore‐like activity, and the observed defect in antibiotic production was found to result from elevated expression of this gene cluster. Promoter regions in the coelibactin cluster contain predicted binding motifs for the zinc‐responsive regulator Zur, and dual regulation of coelibactin expression by zur and absC was demonstrated using strains engineered to contain deletions in either or both of these genes. An AbsC binding site was identified in a divergent promoter region within the coelibactin biosynthetic gene cluster, adjacent to a putative Zur binding site. Repression of the coelibactin gene cluster by both AbsC and Zur appears to be required to maintain appropriate intracellular levels of zinc in S. coelicolor.     

Changes in ponderal index and body mass index across childhood and their associations with fat mass and cardiovascular risk factors at age 15

Background

Little is known about whether associations between childhood adiposity and later adverse cardiovascular health outcomes are driven by tracking of overweight from childhood to adulthood and/or by vascular and metabolic changes from childhood overweight that persist into adulthood. Our objective is to characterise associations between trajectories of adiposity across childhood and a wide range of cardiovascular risk factors measured in adolescence, and explore the extent to which these are mediated by fat mass at age 15.

Methods and Findings

Using data from the Avon Longitudinal Study of Parents and Children, we estimated individual trajectories of ponderal index (PI) from 0–2 years and BMI from 2–10 years using random-effects linear spline models (N = 4601). We explored associations between PI/BMI trajectories and DXA-determined total-body fat-mass and cardiovascular risk factors at 15 years (systolic and diastolic blood pressure, fasting LDL- and HDL-cholesterol, triglycerides, C-reactive protein, glucose, insulin) with and without adjustment for confounders. Changes in PI/BMI during all periods of infancy and childhood were associated with greater DXA-determined fat-mass at age 15. BMI changes in childhood, but not PI changes from 0–2 years, were associated with most cardiovascular risk factors in adolescence; associations tended to be strongest for BMI changes in later childhood (ages 8.5–10), and were largely mediated by fat mass at age 15.

Conclusion

Changes in PI/BMI from 0–10 years were associated with greater fat-mass at age 15. Greater increases in BMI from age 8.5–10 years are most strongly associated with cardiovascular risk factors at age 15, with much of these associations mediated by fat-mass at this age. We found little evidence supporting previous reports that rapid PI changes in infancy are associated with future cardiovascular risk. This study suggests that associations between early overweight and subsequent adverse cardiovascular health are largely due to overweight children tending to remain overweight.     

Eastern spread of the invasive <Emphasis Type="Italic">Artemia franciscana</Emphasis> in the Mediterranean Basin,with the first record from the Balkan Peninsula

Long-term (1987–2015) monitoring data from Lake Lugano (Switzerland/Italy) were used to examine the effects of winter weather, solar radiation and mixing regime on the recovery of the trophic state of a lake undergoing nutrient management. Main hypotheses were that (H1) trends in trophic state were influenced not only by nutrient management, but also by winter weather and solar radiation, and (H2) the effects of management were more pronounced in the holomictic South basin than in the meromictic North basin of the lake. External loadings of phosphorus were strongly reduced during the study period, but key indicators of trophic state, including phosphorous concentration, primary production, chlorophyll a and deep-water oxygenation, showed inconsistent responses. Supporting H1, winter weather (parameterized using an index of the East Atlantic pattern) influenced temporal variation in phosphorus concentration and primary production in the North basin. Supporting H2, the effects of management on trophic state were clearer in the South basin, where most trophic-state indicators declined. Meromixis affected the restoration of the North basin lake by transmitting effects of climatic variation on trophic state. The added variability obscured the effects of restoration and caused sudden deteriorations in water quality, indicating that the restoration of meromictic lakes presents unique challenges.     

Stable isotope probing analysis of the influence of liming on root exudate utilization by soil microorganisms

Rhizosphere microorganisms play an important role in soil carbon flow, through turnover of root exudates, but there is little information on which organisms are actively involved or on the influence of environmental conditions on active communities. In this study, a 13CO2 pulse labelling field experiment was performed in an upland grassland soil, followed by RNA-stable isotope probing (SIP) analysis, to determine the effect of liming on the structure of the rhizosphere microbial community metabolizing root exudates. The lower limit of detection for SIP was determined in soil samples inoculated with a range of concentrations of 13C-labelled Pseudomonas fluorescens and was found to lie between 10(5) and 10(6) cells per gram of soil. The technique was capable of detecting microbial communities actively assimilating root exudates derived from recent photo-assimilate in the field. Denaturing gradient gel electrophoresis (DGGE) profiles of bacteria, archaea and fungi derived from fractions obtained from caesium trifluoroacetate (CsTFA) density gradient ultracentrifugation indicated that active communities in limed soils were more complex than those in unlimed soils and were more active in utilization of recently exuded 13C compounds. In limed soils, the majority of the community detected by standard RNA-DGGE analysis appeared to be utilizing root exudates. In unlimed soils, DGGE profiles from 12C and 13C RNA fractions differed, suggesting that a proportion of the active community was utilizing other sources of organic carbon. These differences may reflect differences in the amount of root exudation under the different conditions.     

Activation tagging using the En-I maize transposon system in Arabidopsis

A method for the generation of stable activation tag inserts was developed in Arabidopsis using the maize (Zea mays) En-I transposon system. The method employs greenhouse selectable marker genes that are useful to efficiently generate large populations of insertions. A population of about 8,300 independent stable activation tag inserts has been produced. Greenhouse-based screens for mutants in a group of plants containing about 2,900 insertions revealed about 31 dominant mutants, suggesting a dominant mutant frequency of about 1%. From the first batch of about 400 stable insertions screened in the greenhouse, four gain-in-function, dominant activation-tagged, morphological mutants were identified. A novel gain-in-function mutant called thread is described, in which the target gene belongs to the same family as the YUCCA flavin-mono-oxygenase that was identified by T-DNA activation tagging. The high frequency of identified gain-in-function mutants in the population suggests that the En-I system described here is an efficient strategy to saturate plant genomes with activation tag inserts. Because only a small number of primary transformants are required to generate an activation tag population, the En-I system appears to be an attractive alternative to study plant species where the present transformation methods have low efficiencies.     

Normal and abnormal heme biosynthesis. Part 7. Synthesis and metabolism of coproporphyrinogen-III analogues with acetate or butyrate side chains on rings C and D. Development of a modified model for the active site of coproporphyrinogen oxidase

Analogues of coproporphyrinogen-III have been prepared with acetate or butyrate groups attached to the C and D pyrrolic subunits. The corresponding porphyrin methyl esters were synthesized by first generating a,c-biladienes by reacting a dipyrrylmethane with pyrrole aldehydes in the presence of HBr. Cyclization with copper(II) chloride in DMF, followed by demetalation with 15% H(2)SO(4)-TFA and reesterification, gave the required porphyrins in excellent yields. Hydrolysis with 25% hydrochloric acid and reduction with sodium-amalgam gave novel diacetate and dibutyrate porphyrinogens 9. Diacetate 9a was incubated with chicken red cell hemolysates (CRH), but gave complex results due to the combined action of two of the enzymes present in these preparations. Separation of uroporphyrinogen decarboxylase (URO-D) from coproporphyrinogen oxidase (CPO) allowed the effects of both enzymes on the diacetate substrate to be assessed. Porphyrinogen 9a proved to be a relatively poor substrate for CPO compared to the natural substrate coproporphyrinogen-III, and only the A ring propionate moiety was processed to a significant extent. Similar results were obtained for incubations of 9a with purified human recombinant CPO. Diacetate 9a was also a substrate for URO-D and a porphyrinogen monoacetate was the major product in this case; however, some conversion of a second acetate unit was also evident. The dibutyrate porphyrinogen 9b was only recognized by the enzyme CPO, but proved to be a modest substrate for incubations with CRH. However, 9b was an excellent substrate for purified human recombinant CPO. The major product for these incubations was a monovinylporphyrinogen, but some divinyl product was also generated in incubations using purified recombinant human CPO. The incubation products were converted into the corresponding porphyrin methyl esters, and these were characterized by proton NMR spectroscopy and mass spectrometry. The results extend our understanding of substrate recognition and catalysis for this intriguing enzyme and have allowed us to extend the active site model for CPO. In addition, the competitive action of both URO-D and CPO on the same diacetate porphyrinogen substrate provides additional perspectives on the potential existence of abnormal pathways for heme biosynthesis.     

NF-κB in the regulation of epithelial homeostasis and inflammation

The IκB kinase/NF-κB signaling pathway has been implicated in the pathogenesis of several inflammatory diseases. Increased activation of NF-κB is often detected in both immune and non-immune cells in tissues affected by chronic inflammation, where it is believed to exert detrimental functions by inducing the expression of proinflammatory mediators that orchestrate and sustain the inflammatory response and cause tissue damage. Thus, increased NF-κB activation is considered an important pathogenic factor in many acute and chronic inflammatory disorders, raising hopes that NF-κB inhibitors could be effective for the treatment of inflammatory diseases. However, ample evidence has accumulated that NF-κB inhibition can also be harmful for the organism, and in some cases trigger the development of inflammation and disease. These findings suggested that NF-κB signaling has important functions for the maintenance of physiological immune homeostasis and for the prevention of inflammatory diseases in many tissues. This beneficial function of NF-κB has been predominantly observed in epithelial cells, indicating that NF-κB signaling has a particularly important role for the maintenance of immune homeostasis in epithelial tissues. It seems therefore that NF-κB displays two faces in chronic inflammation: on the one hand increased and sustained NF-κB activation induces inflammation and tissue damage, but on the other hand inhibition of NF-κB signaling can also disturb immune homeostasis, triggering inflammation and disease. Here, we discuss the mechanisms that control these apparently opposing functions of NF-κB signaling, focusing particularly on the role of NF-κB in the regulation of immune homeostasis and inflammation in the intestine and the skin.     

The identification of a selective dopamine D2 partial agonist,D3 antagonist displaying high levels of brain exposure

The identification of a highly selective D₂ partial agonist, D₃ antagonist tool molecule which demonstrates high levels of brain exposure and selectivity against an extensive range of dopamine, serotonin, adrenergic, histamine, and muscarinic receptors is described.     

Fiber type composition of four hindlimb muscles of adult Fisher 344 rats

 The limb and trunk muscles of adult rats express four myosin heavy chain (MHC) isoforms, one slow (MHCI) and three fast (MHCIIa, MHCIId, and MHCIIb). The distribution of these isoforms correlates with fiber types delineated using myofibrillar actomyosin adenosine triphosphatase (mATPase) histochemistry. For example, type I fibers express MHCI and fiber types IIA, IID, and IIB express MHCIIa, MHCIId, and MHCIIb, respectively. Fibers containing only one MHC isoform have been termed ”pure” fibers. Recent evidence suggests that a population of ”hybrid” fibers exist in rat skeletal muscle which contain two MHC isoforms. The purpose of the present investigation was to document the entire range of histochemically defined ”pure” and ”hybrid” fiber types in untreated muscles of the young adult Fisher 344 rat hindlimb. The selected hindlimb muscles (soleus, tibialis anterior, extensor digitorum longus, and gastrocnemius muscles) were removed from 12 male rats and analyzed for muscle fiber type distribution, cross-sectional area, and MHC content. Care was taken to delineate eight fiber types (I, IC, IIC, IIA, IIAD, IID, IIDB, and IIB) using refined histochemical techniques. Hybrid fibers were found to make up a considerable portion of the muscles examined (a range of 8.8–17.8% of the total). The deep red portion of the gastrocnemius muscle contained the largest number of hybrid fibers, most of which were the fast types IIAD (8.5±2.8%) and IIDB (5.2±2.3%). In conclusion, hybrid fibers make up a considerable portion of normal rat limb musculature and are an important population that should not be ignored. Accepted: 15 October 1998