The netrin receptor frazzled is required in the target for establishment of retinal projections in the Drosophila visual system

Retinal axons in Drosophila make precise topographic connections with their target cells in the optic lobe. Here we investigate the role of the Netrins and their receptor Frazzled in the establishment of retinal projections. We find that the Netrins, although expressed in the target, are not required for retinal projections. Surprisingly, Frazzled, found on both retinal fibers and target cells, is required in the target for attracting retinal fibers, while playing at best a redundant role in the retinal fibers themselves; this finding demonstrates that target attraction is necessary for topographic map formation. Finally, we show that Frazzled is not required for the differentiation of cells in the target. Our data suggest that Frazzled does not function as a Netrin receptor in attracting retinal fibers to the target; nor does it seem to act as a homotypic cell adhesion molecule. We favor the possibility that Frazzled in the target interacts with a component on the surface of retinal fibers, possibly another Netrin receptor.     

Structural analysis of the polysaccharide from the lipopolysaccharide of Porphyromonas gingivalis strain W50.

The lipopolysaccharide (LPS) of Porphyromonas gingivalis is an important pro-inflammatory molecule in periodontal disease and a significant target of the host's specific immune response. In addition, we recently demonstrated using monoclonal antibodies that the Arg-gingipains of P. gingivalis are post-translationally modified with glycan chains that are immunologically related to an LPS preparation from this organism. In the present investigation, we determined the structure of the O-polysaccharide of P. gingivalis W50 that was fully characterized on the basis of 1D and 2D NMR (DQF-COSY, TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-31P HXTOCSY) and GC-MS data. These data allowed us to conclude that the O-polysaccharide is built up of the tetrasaccharide repeating sequence: -->6)-alpha-D-Glcp-(1-->4)-alpha-L-Rhap-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Galp-(1--> and carries a monophosphoethanolamine residue at position C-2 of the alpha-rhamnose residue in a nonstoichiometric (approximately 60%) amount. These data indicate that the O-polysaccharide of P. gingivalis LPS is composed of an unusually modified tetrasaccharide repeating unit.     

Active site characterization of RNase Rs from Rhizopus stolonifer: involvement of histidine and lysine in catalysis and carboxylate in substrate binding.

Chemical modification studies on purified RNase Rs revealed the involvement of a single histidine, lysine and carboxylate residue in the catalytic activity of the enzyme. RNA could not protect the enzyme against DEP- and TNBS-mediated inactivation whereas, substrate protection was observed in case of EDAC-mediated inactivation of the enzyme. K(m) and k(cat) values of the partially inactivated enzyme samples suggested that while histidine and lysine are involved in catalysis, carboxylate is involved in substrate binding. Active site nature of RNase Rs suggests that the inability of the enzyme to readily convert 2',3'-cyclic nucleotides to 3'-mononucleotides is probably due to the absence of catalytically active second histidine residue.     

Platelet-derived growth factor BB mediated regulation of 12-lipoxygenase in porcine aortic smooth muscle cells

Platelet-derived growth factor BB (PDGF) is a potent mitogen and chemoattractant for vascular smooth muscle cells (VSMC). In the present study, we have examined the effects of PDGF on the 12-lipoxygenase (12-LO) pathway of arachidonate metabolism in porcine aortic VSMC (PVSMC). The rationale for this is previous studies showing that LO products have growth and chemotactic effects in VSMC and that another VSMC growth factor, angiotensin II, is a potent positive regulator of 12-LO activity and expression. We observed that PDGF causes a significant increase in the formation of the 12-LO product, 12-hydroxyeicosatetraenoic acid (12-HETE) in PVSMC. In addition, PDGF also markedly increased leukocyte-type 12-LO messenger RNA and protein expression. PDGF-induced PVSMC migration was inhibited significantly by two LO blockers but not by a cyclooxygenase blocker. Furthermore, although the proliferative effects of PDGF on PVSMC were not altered by cell culture under hyperglycemic conditions (25 mM glucose, HG), the chemotactic effects of PDGF as well as those of 10% fetal calf serum were significantly greater in cells cultured in HG as compared to normal glucose conditions (5.5 mM), thus indicating a potential new mechanism for the accelerated cardiovascular disease usually observed in diabetes. These results indicate a novel mechanism for the biological effects of PDGF in leading to cardiovascular disease. © 1996 Wiley-Liss, Inc.     

The development of mouse oocyte cortical reaction competence is accompanied by major changes in cortical vesicles and not cortical granule depth

The basis for the incompetence of the cortical reaction in germinal vesicle stage (GV) mouse oocytes was studied by evaluating cortical granules (CGs) and vesicles in GV and mature oocyte cortices. Dark and light CGs had a similar mean distance of 0.4-0.6 micron from the plasma membrane for GV and mature cortices. The cortex of mature oocytes had a large population of membrane-bounded, 0.1-1.0 micron (diameter) vesicles. More than three times as many vesicles were observed in the CG domains of mature oocytes as were observed in GV oocytes. This lack of cortical vesicles (with their potential to store calcium) and not CG depth may account for cortical reaction incompetence in GV oocytes.     

Yeast cytochrome c is a sequence-specific DNA-binding protein

A protein binding to the alcohol oxidase 2 upstream activation sequence (AOX2UAS) of the methylotropic yeast, Pichia pastoris, has been purified and identified as cytochrome c (cyt c). Cyt c purified from P. pastoris or Saccharomyces cerevisiae binds to AOX2UAS. Specific point mutations in AOX2UAS abolish cyt c binding. We conclude that yeast cyt c is a sequence-specific DNA-binding protein and may have a regulatory role in the nucleus.     

A stromal Hsp100 protein is required for normal chloroplast development and function in Arabidopsis

Molecular chaperones are required for the translocation of many proteins across organellar membranes, presumably by providing energy in the form of ATP hydrolysis for protein movement. In the chloroplast protein import system, a heat shock protein 100 (Hsp100), known as Hsp93, is hypothesized to be the chaperone providing energy for precursor translocation, although there is little direct evidence for this hypothesis. To learn more about the possible function of Hsp93 during protein import into chloroplasts, we isolated knockout mutant lines that contain T-DNA disruptions in either atHSP93-V or atHSP93-III, which encode the two Arabidopsis (Arabidopsis thaliana) homologs of Hsp93. atHsp93-V mutant plants are much smaller and paler than wild-type plants. In addition, mutant chloroplasts contain less thylakoid membrane when compared to the wild type. Plastid protein composition, however, seems to be largely unaffected in atHsp93-V knockout plants. Chloroplasts isolated from the atHsp93-V knockout mutant line are still able to import a variety of precursor proteins, but the rate of import of some of these precursors is significantly reduced. These results indicate that atHsp93-V has an important, but not essential, role in the biogenesis of Arabidopsis chloroplasts. In contrast, knockout mutant plants for atHsp93-III, the second Arabidopsis Hsp93 homolog, had a visible phenotype identical to the wild type, suggesting that atHsp93-III may not play as important a role as atHsp93-V in chloroplast development and/or function.