142 research outputs found
Enabling pandemic speed to clinic for SARS-CoV2 neutralizing antibodies
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Glycomics Analysis of Mammalian Heparan Sulfates Modified by the Human Extracellular Sulfatase HSulf2
The Sulfs are a family of endosulfatases that selectively modify the 6O-sulfation state of cell-surface heparan sulfate (HS) molecules. Sulfs serve as modulators of cell-signaling events because the changes they induce alter the cell surface co-receptor functions of HS chains. A variety of studies have been aimed at understanding how Sulfs modify HS structure, and many of these studies utilize Sulf knockout cell lines as the source for the HS used in the experiments. However, genetic manipulation of Sulfs has been shown to alter the expression levels of HS biosynthetic enzymes, and in these cases an assessment of the fine structural changes induced solely by Sulf enzymatic activity is not possible. Therefore, the present work aims to extend the understanding of substrate specificities of HSulf2 using in vitro experiments to compare HSulf2 activities on HS from different organ tissues.To further the understanding of Sulf enzymatic activity, we conducted in vitro experiments where a variety of mammalian HS substrates were modified by recombinant human Sulf2 (HSulf2). Subsequent to treatment with HSulf2, the HS samples were exhaustively depolymerized and analyzed using size-exclusion liquid chromatography-mass spectrometry (SEC-LC/MS). We found that HSulf2 activity was highly dependent on the structural features of the HS substrate. Additionally, we characterized, for the first time, the activity of HSulf2 on the non-reducing end (NRE) of HS chains. The results indicate that the action pattern of HSulf2 at the NRE is different compared to internally within the HS chain.The results of the present study indicate that the activity of Sulfs is dependent on the unique structural features of the HS populations that they edit. The activity of HSulf2 at HS NREs implicates the Sulfs as key regulators of this region of the chains, and concomitantly, the protein-binding events that occur there
Advances in mass spectrometry-based post-column bioaffinity profiling of mixtures
In the screening of complex mixtures, for example combinatorial libraries, natural extracts, and metabolic incubations, different approaches are used for integrated bioaffinity screening. Four major strategies can be used for screening of bioactive mixtures for protein targets—pre-column and post-column off-line, at-line, and on-line strategies. The focus of this review is on recent developments in post-column on-line screening, and the role of mass spectrometry (MS) in these systems. On-line screening systems integrate separation sciences, mass spectrometry, and biochemical methodology, enabling screening for active compounds in complex mixtures. There are three main variants of on-line MS based bioassays: the mass spectrometer is used for ligand identification only; the mass spectrometer is used for both ligand identification and bioassay readout; or MS detection is conducted in parallel with at-line microfractionation with off-line bioaffinity analysis. On the basis of the different fields of application of on-line screening, the principles are explained and their usefulness in the different fields of drug research is critically evaluated. Furthermore, off-line screening is discussed briefly with the on-line and at-line approaches
Interleukin-1 beta-induced up-regulation of opioid receptors in the untreated and morphine-desensitized U87 MG human astrocytoma cells
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Reduction of ripple voltage in a dynamitron
We determined that a precise neutralization of the RF ripple voltage on the high-voltage terminal of a Dynamitron has previously been prevented by a nonegligible phase shift of RF currents in the two halves of the approx. 100-kHz class C oscillator tank circuit, which is actually constituted of two slightly unequal high-Q coupled circuits because it has two ground points: the inescapable center-tap-ground in the capacitive legs and a center-tap-ground lead to the induction coil. The latter is needed to prevent damage by flashover transients; equivalent to its removal was the adjusting of RF ground return current to a null by aid of a current transformer on this lead and the suitable adjusting of trimmer capacitance. While the phase shift was thus held to a null, the actual ripple amplitude on the hv terminal was minimized by adjusting additional trimmer capacitances installed in the terminal of the machine. Then p/p 100-kHz ripple at 2-MV dc output was reduced to about 50V and RMS resolution by (p,..gamma..) resonance threshold data near 1 MV was about 250 V. The limit to resolution has various causes including mechanical vibrations and unbalanced harmonics of the RF
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