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.     

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.     

Integrated dialysis and renal transplantation: small is beautiful.

Many patients in Britain with chronic renal failure suitable for renal replacement treatment die because not enough treatment facilities are available. Moreover, the number of renal transplants performed is insufficient to meet even present needs, so the number of patients on dialysis is rising. The integrated dialysis and transplant unit in Aberdeen, which has a population base much smaller than the average British unit, meets community needs for dialysis and transplantation. The problem of harvesting cadaver kidneys has been solved; the present supply has not only enabled the number of patients on dialysis to remain stable but has resulted in a net export of kidneys. The Aberdeen unit shows how estimated needs for chronic dialysis and renal transplantation may be met.     

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     

Reverse genetic analysis of the yeast RSC chromatin remodeler reveals a role for RSC3 and SNF5 homolog 1 in ploidy maintenance

The yeast “remodels the structure of chromatin” (RSC) complex is a multi-subunit “switching deficient/sucrose non-fermenting” type ATP-dependent nucleosome remodeler, with human counterparts that are well-established tumor suppressors. Using temperature-inducible degron fusions of all the essential RSC subunits, we set out to map RSC requirement as a function of the mitotic cell cycle. We found that RSC executes essential functions during G1, G2, and mitosis. Remarkably, we observed a doubling of chromosome complements when degron alleles of the RSC subunit SFH1, the yeast hSNF5 tumor suppressor ortholog, and RSC3 were combined. The requirement for simultaneous deregulation of SFH1 and RSC3 to induce these ploidy shifts was eliminated by knockout of the S-phase cyclin CLB5 and by transient depletion of replication origin licensing factor Cdc6p. Further, combination of the degron alleles of SFH1 and RSC3, with deletion alleles of each of the nine Cdc28/Cdk1-associated cyclins, revealed a strong and specific genetic interaction between the S-phase cyclin genes CLB5 and RSC3, indicating a role for Rsc3p in proper S-phase regulation. Taken together, our results implicate RSC in regulation of the G1/S-phase transition and establish a hitherto unanticipated role for RSC-mediated chromatin remodeling in ploidy maintenance.     

SWI-SNF-mediated nucleosome remodeling: role of histone octamer mobility in the persistence of the remodeled state

SWI-SNF is an ATP-dependent chromatin remodeling complex that disrupts DNA-histone interactions. Several studies of SWI-SNF activity on mononucleosome substrates have suggested that remodeling leads to novel, accessible nucleosomes which persist in the absence of continuous ATP hydrolysis. In contrast, we have reported that SWI-SNF-dependent remodeling of nucleosomal arrays is rapidly reversed after removal of ATP. One possibility is that these contrasting results are due to the different assays used; alternatively, the lability of the SWI-SNF-remodeled state might be different on mononucleosomes versus nucleosomal arrays. To investigate these possibilities, we use a coupled SWI-SNF remodeling-restriction enzyme assay to directly compare the remodeling of mononucleosome and nucleosomal array substrates. We find that SWI-SNF action causes a mobilization of histone octamers for both the mononucleosome and nucleosomal array substrates, and these changes in nucleosome positioning persist in the absence of continued ATP hydrolysis or SWI-SNF binding. In the case of mononucleosomes, the histone octamers accumulate at the DNA ends even in the presence of continued ATP hydrolysis. On nucleosomal arrays, SWI-SNF and ATP lead to a more dynamic state where nucleosomes appear to be constantly redistributed and restriction enzyme sites throughout the array have increased accessibility. This random positioning of nucleosomes within the array persists after removal of ATP, but inactivation of SWI-SNF is accompanied by an increased occlusion of many restriction enzyme sites. Our results also indicate that remodeling of mononucleosomes or nucleosomal arrays does not lead to an accumulation of novel nucleosomes that maintain an accessible state in the absence of continuous ATP hydrolysis.