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.     

Effects of sterilization treatments on some properties of alginate solutions and gels.

Aqueous sodium alginate solutions were subjected to various heat sterilization treatments. Sodium alginate powder was also treated by both gamma-irradiation and ethylene oxide sterilization. The effects of these treatments on the viscosities of sodium alginate solutions and both the diameter and strength of the beads formed in 0.1 M CaCl2 solutions were determined quantitatively. The viscosity of sodium alginate solutions and the gel strength of the calcium alginate beads decreased with increasing sterilization temperature while the bead diameters were found to increase. All these effects can be attributable to a reduction in the degree of polymerization of the alginate molecules as a result of the heat treatments. Ethylene oxide and gamma-irradiation treatments caused similar effects. Standard conditions for sterilization are necessary for comparative studies with alginate beads.     

Plasmid stability and ecological competence in recombinant cultures

The instability of cell cultures containing plasmid vectors is a major problem in the commercial exploitation of molecular cloning techniques. Plasmid stability is influenced by the nature of the host cell, the type of plasmid and/or environmental conditions. Plasmid encoded properties may confer a selective advantage on the host cell but can be an energy drain due to replication and expression. Stability of recombinant cultures ultimately may be determined by the cost to benefit ratio of plasmid carriage.The relative competition between plasmid containing and plasmid-free or indigenous populations can determine the degree of dominance of recombinant cultures. The use of inocula in biotechnological processes in which dynamic environmental conditions dominate may also result in instabilities resulting from the characteristics of the ecosystem. In such dynamic conditions plasmid stability is just one contribution to culture stability.Strategies to enhance plasmid stability, within such environments, based on manipulation of physiological state of host cells, must consider the responsiveness or plasticity of both cells and populations. The robustness of cells or the responses to stresses or transient environmental conditions can influence the levels of instability detected; for example, instability or mutation in the host genome may lead to enhanced plasmid stability. Competition among subpopulations arising from unstable copy number control may determine the levels of recombinant cells in open versus closed fermenter systems.Thus the ecological competence (ability to survive and compete) of recombinant cells in dynamic or transient environments is fundamental to the understanding of the ultimate dominance or survival of such recombinant cultures and may form the basis of a strategy to enhance or control stability either in fermenter systems or dynamic process environments. The creation of microniches in time and/or space can enhance plasmid stability. Transient operation based on defined environmental stresses or perturbations in fermenter systems or in heterogeneous or dynamic environments found in gel immobilized cultures have resulted in enhanced stability. Spatial organization resulting from immobilization has the additional advantage of regulated cell protection within defined microenvironments and controlled release, depending on the nature of the gel, from these microenvironments or microcosms. This regulation of ecological competence allied to the advantages of microbial cell growth in gel microenvironments combined with the spatial organization (or juxtapositioning of cells, selective agents, nutrients, protectants, etc.) possible through immobilization technology offers new strategies to enhance plasmid and culture stability.     

Adriamycin effects on transplasma membrane redox functions in porcine neutrophils

Transplasma membrane electron transport, as assayed by external ferricyanide reduction, has been related to control of growth and hormone response of cells. Elicitor-stimulated transmembrane NADPH oxidase is important for bacteriocidal superoxide production by neutrophils. Since adriamycin is myelosuppressive and can stimulate superoxide production, its effects on the two redox systems of porcine neutrophil plasma membranes were compared. Adriamycin inhibits transplasma membrane ferricyanide and stimulates superoxide production activated by phorbol myristate acetate (PMA). Ferricyanide reduction in PMA-treated cells becomes resistant to inhibition by adriamycin. These results provide evidence for an independent effect of adriamycin on transmembrane ferricyanide reduction and on superoxide generation.     

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.     

Malate dehydrogenase. Kinetic studies with meso-tartrate and 2-keto-3-hydroxysuccinate, comparison of the mitochondrial and supernatant pig heart enzymes.

Initial rate, product inhibition, and isotope rate kinetic studies of pig heart mitochondrial and supernatant malate dehydrogenases, acting upon the nonphysiological substrates, meso-tartrate and 2-keto-3-hydroxysuccinate, are reported. The measured spontaneous keto-enol equilibrium for 2-keto-3-hydroxysuccinate in 0.05 m Tris-acetate (pH 8.0) at 25 °C favors the enol form, dihydroxyfumarate, with an apparent equilibrium constant of 0.036. The enzyme-catalyzed reaction favors meso-tartrate with an apparent equilibrium constant of 1.25 × 10^?6, M^?1 at pH 8.0. The mechanism apparently remains ordered bi bi for both enzymes when these nonphysiological substrates are used, and the chemical-converting hydride transfer step becomes more rate limiting for both enzymes. This conclusion is supported by $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{V}{}_{\mathrm{<mtext>D</mtext>}}$ and $\text{(}\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{K}{}_{\mathrm{<mtext>H</mtext>}}\text{)}\text{V}{}_{\mathrm{<mtext>D</mtext>}}\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ values of 2.6 and 3.1, respectively, for the mitochondrial enzyme and 1.9 and 2.9, respectively, for the supernatant enzyme.     

Persistence of introduced Bradyrhizobium japonicum strains in forming nodules in subsequent years after inoculation in Wisconsin soils

Nodulation of soybeans by indigenous and inoculum strains of Bradyrhizobium japonicum was studied in field experiments in Wisconsin from 1983 to 86. Aqueous suspensions of bacteria were applied to seeds at the time of planting at levels of 7?×?10(7)-10(10) bacteria per 2.5-cm row. The predominant indigenous serogroup was 123 in these soils. Six different inoculum strains were used (two from serocluster 123, two from serogroup 110, and one each from serogroups 122 and C1). Nodule occupants were identified using spontaneous antibiotic-resistant mutations in the inoculum strains, phage typing, and serotyping. In the 1983 experiment, the majority of nodules were formed by the inoculum strains in almost all cases (up to 100% in some cases), in two different soils containing 3.5?×?10(5) indigenous B. japonicum per gram. After 2 years without inoculation at the same two site, the inoculum strains did not form many nodules on uninoculated soybeans (less than 10% in most cases; less than 30% in all cases). In inoculation experiments carried out in 1985 and 1986, four inoculum strains were used (3 members of 123 serocluster and USDA 110str); inocula containing 10(8) bacteria per 2.5-cm row formed less than42%ofthe nodules in soils containing 1?×?10(4)-4?×?10(4)B. japonicum per gram. The major conclusions are (i) the success of inoculation in Midwestern U.S. soils is highly variable, even with members of the (highly competitive) 123 serocluster, and (ii) successful inoculation in 1 year in a Wisconsin soil does not ensure that the inoculated strain will persist in forming nodules in that field in subsequent years without further inoculation. Key words: Bradyrhizobium japonicum, strain persistence, field trials.     

Detergent effects upon the isozymes of cytoplasmic glycerol 3-phosphate dehydrogenase

Aminolevulinic acid (ALA) synthase activity was measured in fat body mitochondria from adult male Blaberus discoidalis cockroaches. The enzyme reached its maximum activity at 4 to 6 days of adult age and then dropped to a minimal level which was maintained throughout the remainder of the study period. ALA synthase activity was doubled by allylisopropylacetamide and showed a half-life of about 6 h at 25 °C. Enzyme activity was depressed by long-term allatectomy. However, juvenile hormone administration in vivo did not significantly stimulate the enzyme relative to appropriate controls, and endocrine regulation of fat body ALA synthase remains inconclusive. Hemin inhibited ALA synthase activity, suggesting that fat body heme synthesis could be regulated by end-product inhibition.     

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