Interactions between nutrient availability and hydroperiod shape macroinvertebrate communities in Florida Everglades marshes

Hydroperiod and nutrient status are known to influence aquatic communities in wetlands, but their joint effects are not well explored. I sampled floating periphyton mat and flocculent detritus (floc) infaunal communities using 6-cm diameter cores at short- and long-hydroperiod and constantly inundated sites across a range of phosphorus (P) availability (total phosphorus in soil, floc and periphyton). Differences in community structure between periphyton and floc microhabitats were greater than any variation attributable to hydroperiod, P availability, or other spatial factors. Multivariate analyses indicated community structure of benthic-floc infauna was driven by hydroperiod, although crowding (no. g⁻¹ AFDM) of individual taxa showed no consistent responses to hydroperiod or P availability. In contrast, community structure of periphyton mat infauna was driven by P availability, while densities of mat infauna (no. m⁻²) were most influenced by hydroperiod (+correlations). Crowding of mat infauna increased significantly with P availability in short-hydroperiod marshes, but was constant across the P gradient in long-hydroperiod marshes. Increased abundance of floating-periphyton mat infauna with P availability at short-hydroperiod sites may result from a release from predation by small fish. Community structure and density were not different between long-hydroperiod and constantly inundated sites. These results have implications for the use of macroinvertebrates as indicators of water quality in wetlands and suggest the substrate sampled can influence interpretation of ecological responses observed in these communities.     

Cyclooxygenase blockade attenuates responses of group III and IV muscle afferents to dynamic exercise in cats

Cyclooxygenase products accumulate in statically contracting muscles to stimulate group III and IV afferents. The role played by these products in stimulating thin fiber muscle afferents during dynamic exercise is unknown. Therefore, in decerebrated cats, we recorded the responses of 17 group III and 12 group IV triceps surae muscle afferents to dynamic exercise, evoked by stimulation of the mesencephalic locomotor region. Each afferent was tested while the muscles were freely perfused and while the circulation to the muscles was occluded. The increases in group III and IV afferent activity during dynamic exercise while the circulation to the muscles was occluded were greater than those during exercise while the muscles were freely perfused (P < 0.01). Indomethacin (5 mg/kg iv), a cyclooxygenase blocker, reduced the responses to dynamic exercise of the group III afferents by 42% when the circulation to the triceps surae muscles was occluded (P < 0.001) and by 29% when the circulation was not occluded (P = 0.004). Likewise, indomethacin reduced the responses to dynamic exercise of group IV afferents by 34% when the circulation was occluded (P < 0.001) and by 18% when the circulation was not occluded (P = 0.026). Before indomethacin, the activity of the group IV, but not group III, afferents was significantly higher during postexercise circulatory occlusion than during rest (P < 0.05). After indomethacin, however, group IV activity during postexercise circulatory occlusion was not significantly different from group IV activity during rest. Our data suggest that cyclooxygenase products play a role both in sensitizing group III and IV afferents during exercise and in stimulating group IV afferents during postexercise circulatory occlusion.     

Application of bis(diphenylphosphino)ethane (DPPE) in Staudinger-type N-glycopyranosyl amide synthesis

Bis(diphenylphosphino)ethane (DPPE) reacts with pyranosyl azides derived from D-glucose and D-glucuronic acid in the presence of acid chlorides to yield the corresponding glycosyl amides. Reaction rates are comparable to those with triphenylphosphine, however, the byproduct phosphine oxide is easily removed from reaction mixtures using column chromatography. The simple and clean workup allows for the formation of collections of related compounds by parallel synthesis, and the method is also applicable to scaled-up reactions. The beta-stereochemistry of the glycosyl azide precursor is retained in all cases, which is supported by X-ray crystallography in several cases.     

Compensatory responses to pyruvate carboxylase suppression in islet beta-cells. Preservation of glucose-stimulated insulin secretion

We have previously reported that glucose-stimulated insulin secretion (GSIS) is tightly correlated with pyruvate carboxylase (PC)-catalyzed anaplerotic flux into the tricarboxylic acid cycle and stimulation of pyruvate cycling activity. To further evaluate the role of PC in beta-cell function, we constructed a recombinant adenovirus containing a small interfering RNA (siRNA) specific to PC (Ad-siPC). Ad-siPC reduced PC mRNA levels by 83 and 64% and PC protein by 56 and 35% in INS-1-derived 832/13 cells and primary rat islets, respectively. Surprisingly, this manipulation did not impair GSIS in rat islets. In Ad-siPC-treated 832/13 cells, GSIS was slightly increased, whereas glycolytic rate and glucose oxidation were unaffected. Flux through PC at high glucose was decreased by only 20%, suggesting an increase in PC-specific activity. Acetyl carnitine, a surrogate for acetyl-CoA, an allosteric activator of PC, was increased by 36% in Ad-siPC-treated cells, suggesting a mechanism by which PC enzymatic activity is maintained with suppressed PC protein levels. In addition, the NADPH:NADP ratio, a proposed coupling factor for GSIS, was unaffected in Ad-siPC-treated cells. We conclude that beta-cells activate compensatory mechanisms in response to suppression of PC expression that prevent impairment of anaplerosis, pyruvate cycling, NAPDH production, and GSIS.     

Type Igamma661 phosphatidylinositol phosphate kinase directly interacts with AP2 and regulates endocytosis

Clathrin-coated vesicles mediate sorting and intracellular transport of membrane-bound proteins. The formation of these coats is initiated by the assembly of adaptor proteins (AP), which specifically bind to membrane cargo proteins via recognition of endocytic sorting motifs. The lipid signaling molecule phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is critical for this process, as it serves as both a targeting and regulatory factor. PI(4,5)P(2) is synthesized by type I phosphatidylinositol phosphate kinases (PIPKI). We have discovered a direct interaction between the mu2-subunit of the AP2 complex and PIPKIgamma661 via a yeast two-hybrid screen. This interaction was confirmed using both the mu2-subunit in glutathione S-transferase pulldowns and via coimmunoprecipitation of endogenous PIPKIgamma661 with the AP2 complex from HEK293 cells. The interaction is mediated, in vivo, by a tyrosine-based motif in the 26-amino acid tail of PIPKIgamma661. Because AP2 regulates endocytosis of transferrin receptor from the plasma membrane, we also examined a role for PIPKIgamma661 using a flow cytometry endocytosis assay. We observed that stable expression of wild type PIPKIgamma661 in Madin-Darby canine kidney cells enhanced transferrin uptake, whereas stable expression of kinase-dead PIPKIgamma661 had an inhibitory effect. Neither condition affected the overall cellular level of PI(4,5)P(2). RNA interference-based knockdown of PIPKIgamma661 in HeLa cells also had an inhibitory effect on transferrin endocytosis using the same assay system. Collectively, this evidence implies an important role for PIPKIgamma661 in the AP2-mediated endocytosis of transferrin.     

Anopheles gambiae genome reannotation through synthesis of ab initio and comparative gene prediction algorithms

Background

Complete genome annotation is a necessary tool as Anopheles gambiae researchers probe the biology of this potent malaria vector.

Results

We reannotate the A. gambiae genome by synthesizing comparative and ab initio sets of predicted coding sequences (CDSs) into a single set using an exon-gene-union algorithm followed by an open-reading-frame-selection algorithm. The reannotation predicts 20,970 CDSs supported by at least two lines of evidence, and it lowers the proportion of CDSs lacking start and/or stop codons to only approximately 4%. The reannotated CDS set includes a set of 4,681 novel CDSs not represented in the Ensembl annotation but with EST support, and another set of 4,031 Ensembl-supported genes that undergo major structural and, therefore, probably functional changes in the reannotated set. The quality and accuracy of the reannotation was assessed by comparison with end sequences from 20,249 full-length cDNA clones, and evaluation of mass spectrometry peptide hit rates from an A. gambiae shotgun proteomic dataset confirms that the reannotated CDSs offer a high quality protein database for proteomics. We provide a functional proteomics annotation, ReAnoXcel, obtained by analysis of the new CDSs through the AnoXcel pipeline, which allows functional comparisons of the CDS sets within the same bioinformatic platform. CDS data are available for download.

Conclusion

Comprehensive A. gambiae genome reannotation is achieved through a combination of comparative and ab initio gene prediction algorithms.     

Syk and Slp-76 mutant mice reveal a cell-autonomous hematopoietic cell contribution to vascular development

Developmental studies support a common origin for blood and endothelial cells, while studies of adult angiogenic responses suggest that the hematopoietic system can be a source of endothelial cells later in life. Whether hematopoietic tissue is a source of endothelial cells during normal vascular development is unknown. Mouse embryos lacking the signaling proteins Syk and Slp-76 develop abnormal blood-lymphatic endothelial connections. Here we demonstrate that expression of GFPSlp-76 in a subset of hematopoietic cells rescues this phenotype, and that deficient cells confer focal vascular phenotypes in chimeric embryos consistent with a cell-autonomous mechanism. Endogenous Syk and Slp-76, as well as transgenic GFPSlp-76, are expressed in circulating cells previously proposed to be endothelial precursors, supporting a causal role for these cells. These studies provide genetic evidence for hematopoietic contribution to vascular development and suggest that hematopoietic tissue can provide a source of vascular endothelial progenitor cells throughout life.     

Population patch clamp improves data consistency and success rates in the measurement of ionic currents

Present whole-cell patch-clamp methodology has only moderate consistency and throughput, rendering impractical functional measurements on large numbers of ion channel ligands or on large numbers of unknown or mutant channel genes. In the population patch clamp (PPC) described herein, a single voltage-clamp amplifier sums the whole-cell currents from multiple cells at once, each sealed to a separate aperture in a planar substrate well. The resulting ensemble currents are more consistent from well to well, and the success rate for each recording attempt is >95%. The PPC was implemented by modifying the PatchPlate substrate and amplifiers in the IonWorks patch-clamp instrument. The increased data consistency and likelihood of a successful recording in each well, combined with 384-well measurements in parallel, allow the direct electrophysiological recording of thousands of ensemble ionic currents per day. Therapeutic groups in drug discovery programs require this order of throughput to screen directed compound libraries against ion channel targets. The potential for studying the function of large numbers of ion channel mutants may be realized with the technique. The procedure incorporates subtraction methods that correct for expected distortions and also reliably produces data that agree with previous patch-clamp studies.