Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Vectors for P element-mediated gene transfer in Drosophila.

We have constructed and tested several new vectors for P element-mediated gene transfer. These vectors contain restriction sites for cloning a wide variety of DNA fragments within a small, non-autonomous P element and can be used to efficiently transduce microinjected DNA sequences into the germ line chromosomes of D. melanogaster. The P element in one vector also carries the rosy gene which serves as an easily scored marker to facilitate the transfer of DNA fragments that do not themselves confer a recognizable phenotype. The failure of certain P element constructs to function as vectors suggests that P element sequences, in addition to the 31 bp inverse terminal repeats, are required in cis for transposition. Moreover, removal of the first 38 bp of the autonomous 2.9 kb P element appears to destroy its ability to provide a trans-acting factor (s) required for the transposition of non-autonomous P elements. Finally, we describe a genomic sequence arrangement that apparently arose by the transposition of a 54 kb composite P element from a tetramer plasmid.     

Unusual properties of genomic DNA molecules spanning the euchromatic-heterochromatic junction of a Drosophila minichromosome.

While investigating the copy number of minichromosome Dp(1;f)1187 sequences in the polyploid chromosomes of ovarian nurse and follicle cells of Drosophila melanogaster we discovered that restriction fragments spanning the euchromatic-heterochromatic junction of the chromosome and extending into peri-centromeric sequences had the unusual property of being selectively resistant to transfer out of agarose gels during Southern blotting, leading to systematic reductions in Dp1187-specific hybridization signals. This property originated from the peri-centromeric sequences contained on the junction fragments and was persistently associated with Dp1187 DNA, despite attempts to ameliorate the effect by altering experimental protocols. Transfer inhibition was unlikely to be caused by an inherent physical property of repetitive DNA sequences since, in contrast to genomic DNA, cloned restriction fragments spanning the euchromatic-heterochromatic junction and containing repetitive sequences transferred normally. Finally, the degree of inhibition could be suppressed by the addition of a Y chromosome to the genotype. On the basis of these observations and the fact that peri-centromeric regions of most eukaryotic chromosomes are associated with cytologically and genetically defined heterochromatin, we propose that peri-centromeric sequences of Dp1187 that are incorporated into heterochromatin in vivo retain some component of heterochromatic structure during DNA isolation, perhaps a tightly bound protein or DNA modification, which subsequently causes the unorthodox properties observed in vitro.     

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

Polyploidization and Cell Fusion Contribute to Wound Healing in the Adult Drosophila Epithelium

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The diageotropica mutation alters auxin induction of a subset of the Aux/IAA gene family in tomato

The diageotropica (dgt) mutation has been proposed to affect either auxin perception or responsiveness in tomato plants. It has previously been demonstrated that the expression of one member of the Aux/IAA family of auxin-regulated genes is reduced in dgt plants. Here, we report the cloning of ten new members of the tomato Aux/IAA family by PCR amplification based on conserved protein domains. All of the gene family members except one (LeIAA7) are expressed in etiolated tomato seedlings, although they demonstrate tissue specificity (e.g. increased expression in hypocotyls vs. roots) within the seedling. The wild-type auxin-response characteristics of the expression of these tomato LeIAA genes are similar to those previously described for Aux/IAA family members in Arabidopsis. In dgt seedlings, auxin stimulation of gene expression was reduced in only a subset of LeIAA genes (LeIAA5, 8, 10, and 11), with the greatest reduction associated with those genes with the strongest wild-type response to auxin. The remaining LeIAA genes tested exhibited essentially the same induction levels in response to the hormone in both dgt and wild-type hypocotyls. These results confirm that dgt plants can perceive auxin and suggest that a specific step in early auxin signal transduction is disrupted by the dgt mutation.