AGAP1, an endosome-associated,phosphoinositide-dependent ADP-ribosylation factor GTPase-activating protein that affects actin cytoskeleton

We have identified three members of the AGAP subfamily of ASAP family ADP-ribosylation factor GTPase-activating proteins (Arf GAPs). In addition to the Arf GAP domain, these proteins contain GTP-binding protein-like, ankyrin repeat and pleckstrin homology domains. Here, we have characterized the ubiquitously expressed AGAP1/KIAA1099. AGAP1 had Arf GAP activity toward Arf1>Arf5>Arf6. Phosphatidylinositol 4,5-bisphosphate and phosphatidic acid synergistically stimulated GAP activity. As found for other ASAP family Arf GAPs, the pleckstrin homology domain was necessary for activity. Deletion of the GTP-binding protein-like domain affected lipid dependence of Arf GAP activity. In vivo effects of AGAP1 were distinct from other ASAP family Arf GAPs. Overexpressed AGAP1 induced the formation of and was associated with punctate structures containing the endocytic markers transferrin and Rab4. AP1 was redistributed from the trans-Golgi to the punctate structures. Like other ASAP family members, AGAP1 overexpression inhibited the formation of PDGF-induced ruffles. However, distinct from other ASAP family members, AGAP1 also induced the loss of actin stress fibers. Thus, AGAP1 is a phosphoinositide-dependent Arf GAP that impacts both the endocytic compartment and actin.     

Candidatus "Scalindua brodae", sp. nov., Candidatus "Scalindua wagneri", sp. nov., two new species of anaerobic ammonium oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is both a promising process in wastewater treatment and a long overlooked microbial physiology that can contribute significantly to biological nitrogen cycling in the world's oceans. Anammox is mediated by a monophyletic group of bacteria that branches deeply in the Planctomycetales. Here we describe a new genus and species of anaerobic ammonium oxidizing planctomycetes, discovered in a wastewater treatment plant (wwtp) treating landfill leachate in Pitsea, UK. The biomass from this wwtp showed high anammox activity (5.0 +/- 0.5 nmol/mg protein/min) and produced hydrazine from hydroxylamine, one of the unique features of anammox bacteria. Eight new planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass. Four of these were affiliated to known anammox 16S rRNA gene sequences, but branched much closer to the root of the planctomycete line of descent. Fluorescence in situ hybridization (FISH) with oligonucleotide probes specific for these new sequences showed that two species (belonging to the same genus) together made up > 99% of the planctomycete population which constituted 20% of the total microbial community. The identification of these organisms as typical anammox bacteria was confirmed with electron microscopy and lipid analysis. The new species, provisionally named Candidatus "Scalindua brodae" and "Scalindua wagneri" considerably extend the biodiversity of the anammox lineage on the 16S rRNA gene level, but otherwise resemble known anammox bacteria. Simultaneously, another new species of the same genus, Candidatus "Scalindua sorokinii", was detected in the water column of the Black Sea, making this genus the most widespread of all anammox bacteria described so far.     

Detection and quantification of Plectosphaerella cucumerina,a potential biological control agent of potato cyst nematodes,by using conventional PCR,real-time PCR,selective media,and baiting

Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community. The nematophagous fungus Plectosphaerella cucumerina has demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host. A range of methods have therefore been developed to monitor P. cucumerina. A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence of P. cucumerina in soil, root, and nematode samples. PCR was combined with a bait method to identify P. cucumerina from infected nematode eggs, confirming the parasitic ability of the fungus. A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites. A second P. cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil. PCR, bait, and culture methods were combined to investigate the presence and abundance of P. cucumerina from two field sites in the United Kingdom where PCN populations were naturally declining. All methods enabled differences in the activity of P. cucumerina to be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.     

A new HPLC-ELSD method to quantify indican in Polygonum tinctorium L. and to evaluate beta-glucosidase hydrolysis of indican for indigo production

A method to quantify the indigo precursor indican (indoxyl-beta-D-glucoside) in Polygonum tinctorium L. has been developed. Plant material was extracted in deionized water, and indican was identified and quantified using high performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD). Results confirmed that with this method it is possible to measure indican content in a short time, obtaining reliable and reproducible data. Using this method, leaf indican content was quantified every 15 days during the growing season (from May to October) in P. tinctorium crops grown in a field experiment in Central Italy. Results showed that indican increased along the growing season until flowering and was positively affected by photosynthetic active radiation (PAR). Indican is naturally hydrolyzed by native beta-glucosidase to indoxyl and glucose, the indoxyl yielding indigo. The activity of two enzymes, sweet almond beta-glucosidase and Novarom G preparation, were compared with P. tinctorium native beta-glucosidase to evaluate indigo production. Results showed that the ability to promote indigo formation increased as follows: almond beta-glucosidase 相似文献   

YAC rescue of downless locus mutations in mice

Mice with mutations at the downless (dl) locus have defects in hair follicle, tooth, sweat gland, preputial gland, Meibomian gland, and tail development. The dl phenotype is analogous to the human genetic disorder termed autosomal hypohidrotic (or anhidrotic) ectodermal dysplasia (HED). On the basis of the identification of two related transgenic insertional mutations in the downless gene, yeast artificial chromosomes (YACs) were identified that map to the critical region of mouse Chromosome (Chr) 10. To determine which of the YACs contain the dl gene, we generated YAC transgenic mice by mouse embryo microinjections. The 200-kb YAC B25.D9 was found to rescue all of the downless defects. In addition, the transgenic YAC rescued the dominant Sleek (Dl ^slk ) allele. Since the sequences within the YAC are entirely deleted in one of the transgenic mutants, our results establish that Sleek encodes a dominant-negative protein whose effects can be reversed by expression of extra copies of the wild-type locus. Received: 26 June 1998 / Accepted: 17 July 1998     

Nitrogen Cycling and Nitric Oxide Emissions in Oil-Impacted Prairie Soils

A remote site in the Tallgrass Prairie Preserve of Oklahoma (The Nature Conservancy) was contaminated with crude oil from a pipeline break and is being bioremediated using landfarming techniques. Landfarming is designed to stimulate microbial-based catabolism of petroleum through combined dilution/mixing and fertilization-based effects. To evaluate nitrogen-based effects during remediation, the site was sectioned and treated with urea, ammonium sulfate, or ammonium nitrate. Samples were obtained from prairie soil without chemical nitrogen addition and with or without hydrocarbon contamination. Nitrogen cycling dynamics were followed by measuring ammonium, nitrite, nitrate, and volatile nitric oxide (NO_x) levels. Nitrifying and denitrifying bacterial numbers were estimated and compared to soil oxygen, carbon dioxide, and methane levels as well as to overall total petroleum hydrocarbon (TPH) reduction. For a prairie ecosystem of this type, a high level of fertilization, particularly with nitrogen, can have ecological effects almost as profound as the petroleum contamination itself. Fertilization of the oil-contaminated soil with the reduced and/or oxidized forms of nitrogen quickly resulted in elevated steady-state levels of both ammonium and nitrate, and exceptionally high levels of NO_x released from soil. Although nitrogen fertilization increased microbial nitrogen metabolism and nitrogen cycling, it had minimal effects on the overall remediation efficiency.     

Exercise training reduces skeletal muscle membrane arachidonate in the obese (fa/fa) Zucker rat

Compared with the lean(Fa/) genotype, obese(fa/fa) Zucker rats have arelative deficiency of muscle phospholipid arachidonate, and skeletalmuscle arachidonate in humans is positively correlated with insulinsensitivity. To assess the hypothesis that the positive effects ofexercise training on insulin sensitivity are mediated by increasedmuscle arachidonate, we randomized 20 lean and 20 obese weanling maleZucker rats to sedentary or treadmill exercise groups. After 9 wk,fasting serum, three skeletal muscles (white gastrocnemius, soleus, andextensor digitorum longus), and heart were obtained. Fasting insulinwas halved by exercise training in the obese rat. In whitegastrocnemius and extensor digitorum longus (fast-twitch muscles), butnot in soleus (a slow-twitch muscle) or heart, phospholipidarachidonate was lower in obese than in lean rats(P < 0.001). In all muscles,exercise in the obese rats reduced arachidonate(P < 0.03, by ANOVA contrast). Weconclude that improved insulin sensitivity with exercise in the obesegenotype is not mediated by increased muscle arachidonate and thatreduced muscle arachidonate in obese Zucker rats is unique tofast-twitch muscles.

     

Influence of light on natural indigo production from woad (Isatis tinctoria)

In woad (Isatis tinctoria), field observations indicated, that after periods of dry sunny weather, indigo yields increased significantly, suggesting that light intensity and quality affected indigo precursor production. Therefore, woad was grown under different light intensities and in the presence or absence of supplementary UV light. In general, plants supplied with more light produced more indigo than those given lesser light. When plants under greater light regimes were transferred to lesser light conditions, then indigo production declined. The opposite was also true, indicating that greater indigo production can be obtained from plants harvested after periods of increased sunlight.