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.     

Oral Ultrastructure and Oral Development of the Misaligned Undulating Membrane Mutant of Tetrahymena thermophila1

The misaligned undulating membrane (mum) mutant of Tetrahymena thermophila is a non-conditional, single gene recessive mutation. The major effect of the mum mutation is the production of multiple undulating membrane (UM) fragments in the oral apparatus (OA). The ultrastructure of the UM fragments of mum OAs is identical to that of the single UM of wild-type OAs. Analysis of OA development at midbody using a combination of light microscopy of protargol-stained cells and SEM of demembranated whole cells showed that the phenotypic effect of the mum mutation first becomes evident during mid to late stage 4 and is fully manifested in early stage 5. The effect of the mutation involves a proliferation of excess basal bodies in the UM field. Subsequent events in the development of the mum OA from mid to late stage 5 are identical to those in wild-type OAs. This study suggests that the mum mutation establishes conditions that allow the production of multiple UMs and thus reveals that the UM field is competent for the complete and coordinated development of several adjacent UMs. This level of regional control is not clearly evident when a single UM is present. The comparison of development of wild-type and mum OAs required an extensive reanalysis of stages 4 and 5 of normal oral development. On the basis of current and previous observations, we propose a new and more subdivided staging system for oral development in Tetrahymena.     

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.     

Local and spatially coordinated movements in Dictyostelium discoideum amoebae during chemotaxis

We have studied chemotaxis by individual Dictyostelium discoideum amoebae using strong, local gradients of the chemoattractant cyclic AMP. Gradients were provided by diffusion of cyclic AMP from a microneedle, which could be positioned at various points around the cell. Responses to changes in the gradient indicate how the cell is structurally organized for chemotactic movement. There is a polarity in the responsiveness of the surface to stimulation by cyclic AMP along the length of the amoeba. Furthermore, two aspects of chemotactic movement can be distinguished. The first response to cyclic AMP is a locally generated extension of a hyaline pseudopod from the region of the surface nearest the stimulus. The second response, the flow of cytoplasm in the direction of the stimulus, is coordinated and separate from the first response. The coordination appears to depend on the nucleus or on the microtubule-organizing center.     

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     

Solid-state NMR investigation of the buried X-proline peptide bonds of bacteriorhodopsin

The role of proline residues in the photocycle of bacteriorhodopsin (bR) is addressed using solid-state NMR. (13)C and (15)N chemical shifts from X-Pro peptide bonds in bR are assigned from REDOR difference spectra of pairwise labeled samples, and correlations of chemical shifts with structure are explored in a series of X-Pro model compounds. Results for the three membrane-embedded X-Pro bonds of bR indicate only slight changes in the transition from the resting state of the protein to either the early or late M state of the protonmotive photocycle. These results suggest that the buried prolines serve a principally structural role in bR.     

Expression and purification of active recombinant ATM protein from transiently transfected mammalian cells

The gene mutated in the human disease ataxia telangiectasia (AT), termed ATM, encodes a large protein kinase involved in DNA repair and cell cycle control. Biochemical characterization of ATM function has been somewhat difficult because of its large size (approximately 370 kDa) and relatively low level of expression in several systems. The majority of studies have used immunoprecipitated ATM or purified ATM obtained through relatively complex procedures. Here, we describe an efficient method for the expression and purification of FLAG-epitope-tagged recombinant human ATM protein (F-ATM). This method utilizes the expression of F-ATM in transiently transfected 293T cells followed by anti-FLAG-agarose affinity chromatography. The transfection procedure has been optimized for large (225-cm(2)) culture flasks and F-ATM can be purified to near homogeneity as judged by SDS-PAGE. This procedure yields approximately 1 microg of catalytically active F-ATM protein/225-cm(2) flask that can be used for biochemical studies.     

Variation in the expression of Hsp70, the major heat-shock protein, and thermotolerance in larval and adult selection lines of Drosophila melanogaster

(1) Lines of Drosophila melanogaster were “laboratory naturally” or artificially selected under five thermal regimes. (2) Hsp70 expression per unit protein after heat hardening and heat-shock resistance with and without prior hardening were measured. (3) Differences between the selection regimes in the responses of these traits suggest that thermal resistance can be changed independently of inducible Hsp70 expression. (4) Adult males had higher survival than females but did not differ in inducible Hsp70 expression per unit protein after heat hardening. (5) Larvae expressed less Hsp70 per unit protein than adults after heat hardening.