The Deuterator: software for the determination of backbone amide deuterium levels from H/D exchange MS data

Background  

The combination of mass spectrometry and solution phase amide hydrogen/deuterium exchange (H/D exchange) experiments is an effective method for characterizing protein dynamics, and protein-protein or protein-ligand interactions. Despite methodological advancements and improvements in instrumentation and automation, data analysis and display remains a tedious process. The factors that contribute to this bottleneck are the large number of data points produced in a typical experiment, each requiring manual curation and validation, and then calculation of the level of backbone amide exchange. Tools have become available that address some of these issues, but lack sufficient integration, functionality, and accessibility required to address the needs of the H/D exchange community. To date there is no software for the analysis of H/D exchange data that comprehensively addresses these issues.     

Sperm surface galactosyltransferase activities during in vitro capacitation

Studies using genetic and biochemical probes have suggested that mouse sperm surface galactosyltransferases may participate during fertilization by binding N- acetylglucosamine (GlcNAc) residues in the egg zona pellucida. In light of these results, we examined sperm surface galactosyltransferase activity during in vitro capacitation to determine whether changes in enzymatic activity correlated with fertilizing ability. Results show that surface galactosyltransferases on uncapacitated sperm was preferentially loaded with poly N-acetyllactosamine substrates. As a consequence of capacitation in Ca(++)-containing medium, these polylactosaminyl substrates are spontaneously released from the sperm surface, thereby exposing the sperm galactosyltransferase for binding to the zona pellucida. Sperm capacitation can be mimicked, in the absence of Ca(++), either by washing sperm in Ca(++)-free medium, or by pretreating sperm with antiserum that reacts with the galactosyltransferase substrate. In both instances, sperm galgactosylation of endogenous polylactosaminyl substrates is reduced, coincident with increased galactosylation of exogenous GlcNAc, and increased binding to the zona pellucida. Binding of capacitated sperm to the egg can be inhibited by pronase-digested high molecular weight polyactosaminyl glycoside extracted from epidymal fluids or from undifferentiated F9 embryonal carninoma cells. Thus, these glycosides function as “decapacitation factors” when added back to in vitro fertilization assays. These glycoside “decapacitation factors” inhibit sperm-egg binding by competeing for the sperm surface galactosyltransferase, since (a) they are galactosylated by sperm in the presence of UDP[(3)H]galactose, and (b) enzymatic removal of terminal GlcNAc residues reduces “decapacitation factio” competition. On the other hand “conventional” low molecular weight glycosides, isolated from either epididymal fluid or differentiated F9 cells, fail to inhibit capacitated sperm binding to the zona pellucida. These results define a molecular mechanism for one aspect of sperm capacitation, and help explain why removal of “decapacitation factos” is a necessary prerequisite for sperm binding to the zona pellucida.     

Investigation of the H-mode during ECRH in the T-10 tokamak

An improved confinement regime with an external transport barrier (H-mode) is obtained during electron-cyclotron resonance heating of a plasma in the T-10 tokamak. A characteristic feature of this regime is a spontaneous density growth accompanied by a drop in the intensity of D_α line and an increase in β_p by a factor of ～1.6. The threshold power for the L-H transition is close to that predicted by the ITER scaling. The best characteristics of the H-mode are achieved with decreasing q _L to 2.2. It is shown that the external transport barrier arises for electrons, whereas the heat transport barrier insignificantly contributes to improved confinement.     

Binding of organic anions to a macrocyclic alkaloid d-tubocurarine

An alkaloid d-tubocurarine in aqueous solution binds 8-anilino-1-naphthalenesulfonate and anions of substituted benzoic, aliphatic dicarboxylic, and N-acetyl--amino acids. The binding constants vary from ca. 50 to 3300 M⁻¹ depending on the anion structure, charge and hydrophobicity. The binding of N-acetyl--amino acids is enantiospecific.     

Bcl-2 sensitive mitochondrial potassium accumulation and swelling in apoptosis

During etoposide-induced apoptosis in HL-60 cells, cytochrome c release was associated with mitochondrial swelling caused by increased mitochondrial potassium uptake. The mitochondrial permeability transition was also observed; however, it was not the primary cause of mitochondrial swelling. Potassium uptake and swelling of mitochondria were blocked by bcl-2 overexpression. As a result, cytochrome c release was reduced, and apoptosis delayed. Residual cytochrome c release in the absence of swelling in bcl-2 expressing cells could be due to observed Bax translocation into mitochondria. This study suggests several novel aspects of apoptotic signaling: (1) potassium related swelling of mitochondria; (2) inhibition of mitochondrial potassium uptake by bcl-2; (3) co-existence within one system of multiple mechanisms of cytochrome c release: mitochondrial swelling and swelling-independent permeabilization of the outer mitochondrial membrane.     

Determining the oxidation states of manganese in PC12 and nerve growth factor-induced PC12 cells

Excessive brain Mn can produce toxicity with symptoms resembling parkinsonism. This syndrome, called "manganism," correlates with loss of dopamine in the striatum and cell death in the striatum and globus pallidus. A common hypothesis is that cell damage in Mn toxicity is caused by oxidation of important cell components by Mn3+. Determination of the amount of Mn3+ present, under a range of conditions, in neuronal cells and brain mitochondria represents an important step in evaluating the "damage through oxidation by Mn3+ hypothesis." In an earlier paper we used X-ray absorption near-edge structure (XANES) spectroscopy to determine the amount of Mn2+ and Mn3+ in brain mitochondria under a range of conditions. Here we extend the study to investigate the evidence for formation of Mn3+ through oxidation of Mn2+ by ROS in PC12 cells and in PC12 cells induced with nerve growth factor (NGF) to display a phenotype more like that of neurons. Although the results suggest that very small amounts of Mn3+ might be present at low Mn levels, probably in Mn superoxide dismutase, Mn3+ is not stabilized by complex formation in these cells and therefore does not accumulate to detectable amounts.