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     

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.     

Increased anxiety-like behavior and enhanced synaptic efficacy in the amygdala of GluR5 knockout mice

GABAergic transmission in the amygdala modulates the expression of anxiety. Understanding the interplay between GABAergic transmission and excitatory circuits in the amygdala is, therefore, critical for understanding the neurobiological basis of anxiety. Here, we used a multi-disciplinary approach to demonstrate that GluR5-containing kainate receptors regulate local inhibitory circuits, modulate the excitatory transmission from the basolateral amygdala to the central amygdala, and control behavioral anxiety. Genetic deletion of GluR5 or local injection of a GluR5 antagonist into the basolateral amygdala increases anxiety-like behavior. Activation of GluR5 selectively depolarized inhibitory neurons, thereby increasing GABA release and contributing to tonic GABA current in the basolateral amygdala. The enhanced GABAergic transmission leads to reduced excitatory inputs in the central amygdala. Our results suggest that GluR5 is a key regulator of inhibitory circuits in the amygdala and highlight the potential use of GluR5-specific drugs in the treatment of pathological anxiety.     

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.     

Ring-expanded analogues of natural oxetanocin: (+) and (-) hydroxymethyl isodideoxyadenosine

New enantiomeric isonucleoside analogues related to natural oxetanocin have been synthesized from D-glucosamine and D-glucose. The structures of the target compounds were confirmed by NMR, HRMS, UV, single crystal X-ray, and optical rotation data. Stability studies with respect to purine nucleoside phosphorylase and adenosine deaminase show that these compounds are not substrates. Antiviral results are discussed.     

The species–area relationship in centipedes (Myriapoda: Chilopoda): a comparison between Mediterranean island groups

The present study article examines the shapes of centipede species–area relationships (SARs) in the Mediterranean islands, compares the results of the linear form of the power model between archipelagos, discusses biological significance of the power model parameters with other taxa on the Aegean archipelago, and tests for a significant small‐island effect (SIE). We used 11 models to test the SARs and we compared the quality‐of‐fit of all candidate models. The power function ranked first and Z‐values was in the range 0.106–0.334. We assessed the presence of SIEs by fitting both a continuous and discontinuous breakpoint regression model. The continuous breakpoint regression functions never performed much better than the closest discontinuous model as a predictor of centipede species richness. We suggest that the relatively low Z‐values in our data partly reflect better dispersal abilities in centipedes than in other soil invertebrate taxa. Longer periods of isolation and more recent island formation may explain the somewhat lower constant c in the western Mediterranean islands compared to the Aegean islands. Higher breakpoint values in the western Mediterranean may also be a result of larger distance to the mainland and longer separation times. Despite the differences in the geological history and the idiosyncratic features of the main island groups considered, the overall results are quite similar and this could be assigned to the ability of centipedes to disperse across isolation barriers. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 146–159.     

Selective activation of microglia in spinal cord but not higher cortical regions following nerve injury in adult mouse

Neuronal plasticity along the pathway for sensory transmission including the spinal cord and cortex plays an important role in chronic pain, including inflammatory and neuropathic pain. While recent studies indicate that microglia in the spinal cord are involved in neuropathic pain, a systematic study has not been performed in other regions of the central nervous system (CNS). In the present study, we used heterozygous Cx3cr1 ^GFP/+mice to characterize the morphological phenotypes of microglia following common peroneal nerve (CPN) ligation. We found that microglia showed a uniform distribution throughout the CNS, and peripheral nerve injury selectively activated microglia in the spinal cord dorsal horn and related ventral horn. In contrast, microglia was not activated in supraspinal regions of the CNS, including the anterior cingulate cortex (ACC), prefrontal cortex (PFC), primary and secondary somatosensory cortex (S1 and S2), insular cortex (IC), amygdala, hippocampus, periaqueductal gray (PAG) and rostral ventromedial medulla (RVM). Our results provide strong evidence that nerve injury primarily activates microglia in the spinal cord of adult mice, and pain-related cortical plasticity is likely mediated by neurons.