The sorting nexin, DSH3PX1, connects the axonal guidance receptor, Dscam, to the actin cytoskeleton

Dock, an adaptor protein that functions in Drosophila axonal guidance, consists of three tandem Src homology 3 (SH3) domains preceding an SH2 domain. To develop a better understanding of axonal guidance at the molecular level, we used the SH2 domain of Dock to purify a protein complex from fly S2 cells. Five proteins were obtained in pure form from this protein complex. The largest protein in the complex was identified as Dscam (Down syndrome cell adhesion molecule), which was subsequently shown to play a key role in directing neurons of the fly embryo to correct positions within the nervous system (Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E., and Zipursky, S. L. (2000) Cell 101, 671-684). The smallest protein in this complex (p63) has now been identified. We have named p63 DSH3PX1 because it appears to be the Drosophila orthologue of the human protein known as SH3PX1. DSH3PX1 is comprised of an NH(2)-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. Because of its PX domain, DSH3PX1 is considered to be a member of a growing family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking. We demonstrate that DSH3PX1 immunoprecipitates with Dock and Dscam from S2 cell extracts. The domains responsible for the in vitro interaction between DSH3PX1 and Dock were also identified. We further show that DSH3PX1 interacts with the Drosophila orthologue of Wasp, a protein component of actin polymerization machinery, and that DSH3PX1 co-immunoprecipitates with AP-50, the clathrin-coat adapter protein. This evidence places DSH3PX1 in a complex linking cell surface receptors like Dscam to proteins involved in cytoskeletal rearrangements and/or receptor trafficking.     

Starch synthesis in transgenic potato tubers with increased 3-phosphoglyceric acid content as a consequence of increased 6-phosphofructokinase activity

The aim of this work was to test the hypothesis that changes in cytosolic 3-phosphoglyceric acid (3-PGA) content can regulate the rate of starch synthesis in potato (Solanum tuberosum L.) tubers. The amount of 3-PGA was increased by expressing bacterial phosphofructokinase (PFK; EC 2.7.1.11) in transgenic potato tubers. The resultant 3-fold increase in PFK activity was accompanied by an increase in metabolites downstream of PFK, including a 3-fold increase in 3-PGA. There was also a decrease in metabolites upstream of PFK, most notably of glucose-6-phosphate. The increase in 3-PGA did not affect the amount of starch that accumulated in developing tubers, nor its rate of synthesis in tuber discs cut from developing tubers. This suggests that changes in cytosolic 3-PGA may not affect the rate of starch synthesis under all circumstances. We propose that in this case, a decrease in glucose-6-phosphate (which is transported into the amyloplast as a substrate for starch synthesis) may be sufficient to counteract the effect of increased 3-PGA.     

The role of minor groove functional groups in DNA hydration

Here we describe the crystal structure of modified [d(CGCGAATTCGCG)]₂ refined to 2.04 Å. The modification, which affects only the two thymines at the central ApT step, involves isosteric removal of the 2-keto oxygen atoms and substitution of the N1 nitrogen with carbon. The crystal structure reveals the ability of this modified thymine to effectively base pair with adenine in [d(CGCGAAtTCGCG)]₂. The structure also suggests that the minor groove ‘spine of hydration’ is destabilized but essentially intact.     

Effect of arachidonic acid on duodenal enterocyte ATPases

Duodenal ion transport processes are supported by ATPase enzymes in basolateral membranes of the enterocyte. In vivo studies have shown that long term n-6 poly-unsaturated fatty acid (PUFA) supplementation in rats causes increases in intestinal Ca absorption, coupled with a higher total calcium balance and bone calcium content. The present in vitro study was undertaken to test the effect of arachidonic acid (AA), a highly unsaturated (and thus physiologically potent) member of the n-6 PUFA family, on ATPases in enterocyte basolateral membranes isolated with a sorbitol density gradient procedure. This paper presents results which show that AA inhibits Na+,K+-ATPase in a dose-dependent manner (-67% of basal activity at a concentration of 30 microg/ml, P < 0.005) but that this effect is not mediated by protein kinase C, as shown by the use of the protein kinase C blocker calphostin (0.5 microM). Indomethacin (IDM) at 0.1 mM, a cyclo-oxygenase blocker, could also not reverse the inhibitory effect of AA on Na+,K+-ATPase. Ca2+-ATPase, on the other hand, is not affected significantly (-10%, P > 0.05) by arachidonic acid at 30 microg/ml.     

Cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity

Stem cells in different regions of the nervous system give rise to different types of mature cells. This diversity is assumed to arise in response to local environmental differences, but the contribution of cell-intrinsic differences between stem cells has been unclear. At embryonic day (E)14, neural crest stem cells (NCSCs) undergo primarily neurogenesis in the gut but gliogenesis in nerves. Yet gliogenic and neurogenic factors are expressed in both locations. NCSCs isolated by flow-cytometry from E14 sciatic nerve and gut exhibited heritable, cell-intrinsic differences in their responsiveness to lineage determination factors. Gut NCSCs were more responsive to neurogenic factors, while sciatic nerve NCSCs were more responsive to gliogenic factors. Upon transplantation of uncultured NCSCs into developing peripheral nerves in vivo, sciatic nerve NCSCs gave rise only to glia, while gut NCSCs gave rise primarily to neurons. Thus, cell fate in the nerve was stem cell determined.     

Thermoacoustic molecular imaging of small animals

We have designed, constructed, and tested a thermoacoustic computed tomography (TCT) scanner for imaging optical absorption in small animals in three dimensions. The device utilizes pulsed laser irradiation (680-1064 nm) and a unique, 128-element transducer array. We quantified the isotropic spatial resolution of this scanner to be 0.35 mm. We describe a dual-wavelength subtraction technique for isolating optical dyes with TCT. Phantom experiments demonstrate that we can detect 5 fmol of a near-infrared dye (indocyanine green, ICG) in a 1-microL volume using dual-wavelength subtraction. Initial TCT imaging in phantoms and in two sacrificed mice suggests that three-dimensional, optical absorption patterns in small animals can be detected with an order of magnitude better spatial resolution and an order of magnitude better low-contrast detectability in small animals when compared to fluorescence imaging or diffusion optical tomography.     

Temporally distinct requirements for endothelin receptor B in the generation and migration of gut neural crest stem cells

Loss of Endothelin-3/Endothelin receptor B (EDNRB) signaling leads to aganglionosis of the distal gut (Hirschsprung's disease), but it is unclear whether it is required primarily for neural crest progenitor maintenance or migration. Ednrb-deficient gut neural crest stem cells (NCSCs) were reduced to 40% of wild-type levels by embryonic day 12.5 (E12.5), but no further depletion of NCSCs was subsequently observed. Undifferentiated NCSCs persisted in the proximal guts of Ednrb-deficient rats throughout fetal and postnatal development but exhibited migration defects after E12.5 that prevented distal gut colonization. EDNRB signaling may be required to modulate the response of neural crest progenitors to migratory cues, such as glial cell line-derived neurotrophic factor (GDNF). This migratory defect could be bypassed by transplanting wild-type NCSCs directly into the aganglionic region of the Ednrb(sl/sl) gut, where they engrafted and formed neurons as efficiently as in the wild-type gut.     

Inhibition of duodenal enterocyte Mg2+-ATPase by arachidonic acid is not mediated by an effect on protein kinase C

Active absorption processes in the duodenal enterocyte are driven by various ATPases. It is known that the activity of Na+,K+-ATPase, Ca2+-ATPase and Mg2+-ATPase can be modulated by polyunsaturated fatty acids of the n-6 series, for example by linoleic and gamma-linolenic acids. These effects may be achieved by protein phosphorylation via protein kinase C. The present study was undertaken to determine the effect of arachidonic acid on Mg2+-ATPase (measured colorimetrically) activity in basolateral membranes prepared from rat duodenum. It shows, for the first time, significant dose-dependent inhibition of Mg2+-ATPase (26-62%) by arachidonic acid (10-50 microg/ml) which already takes place after one minute of exposure, indicating involvement of a rapid signal transduction mechanism. Addition of the protein kinase C inhibitors bisimidolylmaleimide (2.5 microM) and calphostin (0.5 microM) did not influence the action of arachidonic acid on Mg2+-ATPase; protein kinase C involvement in this process is thus not indicated.     

Mac-1-negative B-1b phenotype of natural antibody-producing cells, including those responding to Gal alpha 1,3Gal epitopes in alpha 1,3-galactosyltransferase-deficient mice

Human natural Abs against Galalpha1-3Galbeta1-4GlcNAc (Gal) epitopes are a major barrier to xenotransplantation. Studies in this report, which use combined multiparameter flow cytometric sorting and enzyme-linked immunospot assay, demonstrate that anti-Gal IgM-producing cells are found exclusively in a small B cell subpopulation (i.e., CD21(-/low) IgM(high) B220(low) CD5(-) Mac-1(-) 493(-) cells) in the spleens of alpha1, 3-galactosyltransferase-deficient mice. All IgM-producing cells were detected in a similar splenic subpopulation of alpha1, 3-galactosyltransferase-deficient and wild-type mice. A higher frequency of B cells with anti-Gal surface IgM receptors was observed in the peritoneal cavity than in the spleen, but these did not actively secrete Abs, and showed phenotypic properties of B-1b cells (CD21(-/low) IgM(high) CD5(-) CD43(+) Mac-1(+)). However, these became Mac-1(-) and developed anti-Gal Ab-producing activity after in vitro culture with LPS. The splenic B cells with anti-Gal receptors consisted of both Mac-1(+) B-1b cells and Mac-1(-) B-1b-like cells. The latter comprised most anti-Gal IgM-producing cells. Our studies indicate that anti-Gal natural IgM Abs are produced by a B1b-like, Mac-1(-) splenic B cell population and not by plasma cells or B-1a cells. They are consistent with a model whereby B-1b cells lose Mac-1 expression upon Ag exposure and that these, rather than plasma cells, become the major IgM Ab-producing cell population.