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.     

Developmental potential of isolated blastomeres from early murine embryos

Experiments were designed to evaluate the effect of blastomere separation on blastocoele formation and development of viable fetuses. Two-cell and four-cell murine embryos were dissociated into individual blastomeres and cultured to the blastocyst stage. For embryos of both stages, zona removal and blastomere separation reduced (P<0.05) the number of viable embryos at the onset of culture and reduced (P<0.01) the frequency of continuation of development of blastomeres to the blastocyst stage. Attempts to repeatedly split two-cell stage embryos decreased in vitro development to blastocysts. The number of cells in two-cell embryos that were cultured to blastocyst was not different for control (64.8 +/- 11.5) or for two-cell embryos cultured without the zona pellucida (60.9 +/- 10.1) but was reduced (P<0.01) for one-half embryos that were cultured to blastocysts (35.6 +/- 10.6). The cell number of blastocysts obtained from dissociated four-cell (1/4) embryos (17.4 +/- 1.4) was similarly reduced (P<0.01). In vivo development was assessed after cultured embryos were transferred to the uteri of day 3 pseudopregnant females. Zona free intact embryos (2/36, 6%) and zona free half embryos (7/36; 19%) developed less frequently (P<0.05) than intact controls (45/100). Noncultured morula briefly exposed to pronase to thin the zona had similar impaired development. Embryos with thinned zona or no zona developed less frequently (21/82, 2/72 respectively, P<0.05) than nonpronase-treated controls (50/83).     

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     

Hepatocyte susceptibility to glyoxal is dependent on cell thiamin content

Glyoxal, a reactive dicarbonyl, is detoxified primarily by the glyoxalase system utilizing glutathione (GSH) and by the aldo-keto reductase enzymes which utilizes NAD[P]H as the co-factor. Thiamin (Vitamin B(1)) is an essential coenzyme for transketolase (TK) that is part of the pentose phosphate pathway which helps maintain cellular NADPH levels. NADPH plays an intracellular role in regenerating glutathione (GSH) from oxidized GSH (GSSG), thereby increasing the antioxidant defenses of the cell. In this study we have focused on the prevention of glyoxal toxicity by supplementation with thiamin (3mM). Thiamin was cytoprotective and restored NADPH levels, glyoxal detoxification and mitochondrial membrane potential. Hepatocyte reactive oxygen species (ROS) formation, lipid peroxidation and GSH oxidation were decreased. Furthermore, hepatocytes were made thiamin deficient with oxythiamin (3mM) as measured by the decreased hepatocyte TK activity. Under thiamin deficient conditions a non-toxic dose of glyoxal (2mM) became cytotoxic and glyoxal metabolism decreased; while ROS formation, lipid peroxidation and GSH oxidation was increased.     

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.     

Early stages of the apoptotic pathway in plant cells are reversible

Chromatin condensation and nDNA fragmentation, indicators of apoptosis in mammalian cells, occur in plant cells during senescence and following induction by chemical agents. In Nicotiana plumbaginifolia cells, camptothecin, okadaic acid, salicylic acid, hydrogen peroxide, and the calcium ionophore A23187 induced chromatin condensation and nDNA fragmentation. Exposure of cells to low concentrations or removal of the chemical agent resulted in an initial phase of chromatin condensation, followed by its reversal. A further feature of apoptosis in mammalian cells, annexin V binding, indicative of phosphotidylserine exposure, was also confirmed in relation to the other events in the apoptotic pathway. With respect to flow cytometric characteristics, apoptosis triggered by a variety of chemicals occurs in plant cells in a manner closely related to that in mammalian cells. However, the extent of chromatin condensation is substantially greater, and in the early stages is reversible.     

Biochemical isolation and purification of ovulation-inducing factor (OIF) in seminal plasma of llamas

Background  

The objective of the present study was to isolate and purify the protein fraction(s) of llama seminal plasma responsible for the ovulation-inducing effect of the ejaculate.     

Mismatched antigen prepares gamma delta T cells for suppression of airway hyperresponsiveness

Gammadelta T cells suppress airway hyperresponsiveness (AHR) induced in allergen-challenged mice but it is not clear whether the suppression is allergen specific. The AHR-suppressive cells express TCR-Vgamma4. To test whether the suppressive function must be induced, we adoptively transferred purified Vgamma4(+) cells into gammadelta T cell-deficient and OVA-sensitized and -challenged recipients (B6.TCR-Vgamma4(-/-)/6(-/-)) and measured the effect on AHR. Vgamma4(+) gammadelta T cells isolated from naive donors were not AHR-suppressive, but Vgamma4(+) cells from OVA-stimulated donors suppressed AHR. Suppressive Vgamma4(+) cells could be isolated from lung and spleen. Their induction in the spleen required sensitization and challenge. In the lung, their function was induced by airway challenge alone. Induction of the suppressors was associated with their activation but it did not alter their ability to accumulate in the lung. Vgamma4(+) gammadelta T cells preferentially express Vdelta4 and -5 but their AHR-suppressive function was not dependent on these Vdeltas. Donor sensitization and challenge not only with OVA but also with two unrelated allergens (ragweed and BSA) induced Vgamma4(+) cells capable of suppressing AHR in the OVA-hyperresponsive recipients, but the process of sensitization and challenge alone (adjuvant and saline only) was not sufficient to induce suppressor function, and LPS as a component of the allergen was not essential. We conclude that AHR-suppressive Vgamma4(+) gammadelta T cells require induction. They are induced by allergen stimulation, but AHR suppression by these cells does not require their restimulation with the same allergen.