Pole Cell Migration through the Gut Wall of the Drosophila Embryo: Analysis of Cell Interactions

Early in development the precursors of germ cells in Drosophila migrate at the posterior pole of the embryo and translocate to the bottom of the developing posterior midgut primordium. At the end of germ band elongation the pole cells cross the gut wall to enter in association with the gonadal mesoderm. We used laser scanning confocal microscopy on whole-mount Rh-phalloidin-stained embryos and transmission electron microscopy to investigate how pole cells cross the epithelial wall of the posterior midgut primordium. Our results suggest that pole cells leave the midgut sac by traveling through the intercellular spaces of the epithelium. During this process the epithelial cells at the bottom of the posterior midgut primordium are greatly deformed, but their junctional complexes do not completely release, avoiding breaks in the epithelial wall.     

In vitro mimicry of CaMBr1 tumor-associated antigen by synthetic oligosaccharides

The murine monoclonal antibody (Mab) MBr1, raised against thebreast cancer cell line MCF7, recognizes a saccharidic epitopeoverexpressed on a high percentage of human breast, ovary, andlung carcinomas. This antigen was originally identified on theimmunogen as a globo-series glycosphingolipid with an H-likedeterminant at its terminus (globo-H). We report here the biologicalcharacterization of the entire globo-H hexasaccharide and fivesynthetic oligosaccharides representing fragments of the entirestructure andlor different anomeric configurations. Using competitivebinding assays on live cells, we identified the residues andthe linkages essential for mimicry of the cellular antigensrecognized by Mab MBr1 on the breast carcinoma cell line MCF7and small cell lung cancer cell line POVD. The terminal tetrasaccharidicfragment of globo-H is the oligosaccharide that most resemblesthe MBr1-defined epitope both on glycolipids and on glycoproteins.This information will help in the rational design of a highlyspecific reagent for active specific immunotherapy of carcinomasoverexpressing the MBr1-defined antigen. CaMBr1 immunotherapy monoclonal antibody oligosaccharides tumor-associated antigen     

Solution conformation of Lewis a-derived selectin ligands is unaffected by anionic substituents at the 3'- and 6'- positions

The selectins are a family of proteins that mediate leukocytetethering and rolling along the vascular endothelium. E-, P-,and L-selectin recognize various derivatives of the Lewis^a andLewis^x trisaccharides. The distribution of negative chargeson the Lewis^a and Lewis^x oligosaccharides appears to be an importantfactor in their binding by the selectins. Previous work exploringthis electrostatic dependence found that a series of syntheticanionic trisaccharides, 3'-sulfo, 3'-phospho, 6'-sulfo, and3',6'-disulfo Lewis^a. (Glc), exhibited differing selectin inhibitoryefficacies. To explore the possibility that these differencesarise from conformational differences between the sugars, thesolution structures of these trisaccharides were determinedusing NMR and molecular dynamics simulations. Interproton distancesand interglycosidic torsion angles were determined at 37°Cusing NOESY buildup curves and 1D LRJ experiments, respectively.Data from both experiments agreed well with predictions madefrom 2000 picosecond unrestrained molecular dynamics simulations.We found that 3'-sulfation did not alter the core Lewis^a conformation,a finding that reaffirms the results of previous study. In addition,we found that sulfation at the 6' position also leaves the trisaccharideconformation unperturbed. This is significant because the proximityof the 6'-sulfate group to the fucose ring might have alteredthe canonical Lewis^a structure. The disulfate exhibited greaterflexibility than the other derivatives in dynamics simulations,but not so much as to affect NOE and heteronuclear couplingconstant measurements. Taken together, our findings supportthe use of Lewis^a as a template onto which charged groups maybe added without significantly altering the trisaccharide'sstructure. oligosaccharides molecular dynamics simulations NMR sulfated Lewis^a phosphorylated Lewis^a     

Reduction of vanadate to vanadyl by a strain of Saccharomyces cerevisiae

Three strains of Saccharomyces cerevisiae, SC-1, DBVPG 6173 and DBVPG 6037, were studied for vanadate resistance in complex Sabouraud medium since they did not thrive in different minimal media (yeast nitrogen base with and without amino acids). The strain SC-1 was resistant up to 16 mm of vanadate, whereas the strains DBVPG 6173 and DBVPG 6037 were inhibited by 8 mm and 4 mm vanadate, respectively. The vanadate resistance in strain SC-1 was constitutive and due to the reduction of this oxyanion to vanadyl, which was detected by EPR spectroscopy and visible spectroscopy. The transformation of vanadate to vanadyl took place during the exponential growth phase; 10 mm of vanadate was reduced to vanadyl outside the cells since the oxyanion was not detected in the cell biomass and only a negligible concentration of vanadyl (25 nmoles mg cells dry weight) was found in the biomass. The other two vanadate-sensitive yeast strains only accumulated vanadate and did not reduce the oxyanion to vanadyl.     

Clinical phenotype of nephrogenic diabetes insipidus in females heterozygous for a vasopressin type 2 receptor mutation

Nephrogenic diabetes insipidus (NDI) usually shows an X-linked recessive mode of inheritance caused by mutations in the vasopressin type 2 receptor gene (AVPR2). In the present study, three NDI families are described in which females show clinical features resembling the phenotype in males. Maximal urine osmolality in three female patients did not exceed 200 mosmol/kg and the absence of extra-renal responses to 1-desamino-8-d-arginine vasopressin was demonstrated in two of them. All affected females and two asymptomatic female family members were shown to be heterozygous for an AVPR2 mutation. Skewed X-inactivation is the most likely explanation for the clinical manifestation of NDI in female carriers of an AVPR2 mutation. It is concluded that, in female NDI patients, the possibility of heterozygosity for an AVPR2 gene mutation has to be considered in addition to homozygosity for mutations in the aquaporin 2 gene.     

Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs

Summary A general procedure, using the commonly employed solid-phase peptide synthesis methodology for obtaining internally quenched fluorogenic peptides with ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs, is presented. The essential feature of this procedure is the synthesis of an N -Boc or-Fmoc derivative of glutamic acid with the -carboxyl group bound to N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp), which provides the quencher moiety attached to the C-terminus of the substrate. The fluorescent donor group, ortho-aminobenzoic acid (Abz), is incorporated into the resin-bound peptide in the last coupling cycle. Depending on the resin type used, Abz-peptidyl-Gln-EDDnp or Abz-peptidyl-Glu-EDDnp is obtained. Using the procedure described above, substrates for human renin and tissue kallikreins were synthesised. Spectrofluorimetric measurements of Abz bound to the -amino group of proline showed that strong quenching of Abz fluorescence occurs in the absence of any acceptor group.     

ON THE MECHANISM OF ELECTROSHOCK-INDUCED INHIBITION OF PROTEIN SYNTHESIS IN RABBIT CEREBRAL CORTEX

Abstract— The mechanism of electroshock (ES)-induced inhibition of protein synthesis in rabbit cerebral cortex has been investigated by using a cell-free system. The protein biosynthetic activity of the post-mitochondrial supernatant (PMS) obtained from the cerebral cortex of ES-treated animals was found to be markedly lower than in controls (C). This inhibition was accompanied by a decrease of polysomes and an increase of monomers. In addition, a relative increase in light polysomes was evident at short intervals after ES treatment. No difference was found in the total soluble activity and in the activity of the elongation factors and ribonuclease present in the cell sap of C and ES animals. The biosynthetic activity of ES-total. free and membrane-bound ribosomes was approx 45% lower than that of the corresponding C fractions: polysome/monomer ratios were similarly reduced. The total content of cortical ribosomes was not affected by ES. Following ES treatment there was no change in the ribo-somal ability to elongate, terminate and release polypeptide chains, nor a decrease in the polysomal content of poly(A)-containing mRNA. These data strongly suggest that the ES-induced inhibition of protein synthesis results from a defect in the initiation process. The possible mechanisms mediating this defect have been discussed.     

Effect of rye embryo ribosome nuclease on double-stranded RNA

In extracts obtained by treating rye embryo ribosomes with 0.5 M NH₄Cl, nuclease activity was noted towards double-stranded RNA from virus of Penicillium chrysogenum and towards synthetic poly (A)-poly (U) and poly (I)-poly (C) complexes.