Metal-free Superoxide Dismutase-1 and Three Different Amyotrophic Lateral Sclerosis Variants Share a Similar Partially Unfolded ��-Barrel at Physiological Temperature

The structure and unfolding of metal-free (apo) human wild-type SOD1 and three pathogenic variants of SOD1 (A4V, G93R, and H48Q) that cause familial amyotrophic lateral sclerosis have been studied with amide hydrogen/deuterium exchange and mass spectrometry. The results indicate that a significant proportion of each of these proteins exists in solution in a conformation in which some strands of the β-barrel (i.e. β2) are well protected from exchange at physiological temperature (37 °C), whereas other strands (i.e. β3 and β4) appear to be unprotected from hydrogen/deuterium exchange. Moreover, the thermal unfolding of these proteins does not result in the uniform incorporation of deuterium throughout the polypeptide but involves the local unfolding of different residues at different temperatures. Some regions of the proteins (i.e. the “Greek key” loop, residues 104–116) unfold at a significantly higher temperature than other regions (i.e. β3 and β4, residues 21–53). Together, these results show that human wild-type apo-SOD1 and variants have a partially unfolded β-barrel at physiological temperature and unfold non-cooperatively.     

The chloroplast grana proteome defined by intact mass measurements from liquid chromatography mass spectrometry

Proteomics seeks to address the entire complement of protein gene products of an organism, but experimental analysis of such complex mixtures is biased against low abundance and membrane proteins. Electrospray-ionization mass spectrometry coupled with reverse-phase chromatography was used to separate and catalogue all detectable proteins in samples of photosystem II-enriched thylakoid membrane subdomains (grana) from pea and spinach. Around 90 intact mass tags were detected corresponding to approximately 40 gene products with variable post-translational covalent modifications. Provisional identity of 30 of these gene products was proposed based upon coincidence of measured mass with that calculated from genomic sequence. Analysis of isolated photosystem II complexes allowed detection and resolution of a minor population of D1 (PsbA) that was apparently palmitoylated and not detected in less purified preparations. Based upon observed +80-Da adducts, D1, D2 (PsbD), CP43 (PsbC), two Lhcbs, and PsbH were confirmed to be phosphorylated, and a new phosphoprotein was proposed to be the product of psbT. The appearance of a second +80-Da adduct on PsbH provides direct evidence for a second phosphorylation site on PsbH, complicating interpretation of its role in regulation of thylakoid membrane organization and function, including light-state transitions. Adducts of +32 Da, presumably arising from oxidative modification during illumination, were associated with more highly phosphorylated forms of PsbH implying a relationship between the two phenomena. Intact mass proteomics of organellar subfractions and more highly purified protein complexes provides increasingly detailed insights into functional genomics of photosynthetic membranes.     

Mutations in CYC1, Encoding Cytochrome c1 Subunit of Respiratory Chain Complex III,Cause Insulin-Responsive Hyperglycemia

Many individuals with abnormalities of mitochondrial respiratory chain complex III remain genetically undefined. Here, we report mutations (c.288G>T [p.Trp96Cys] and c.643C>T [p.Leu215Phe]) in CYC1, encoding the cytochrome c₁ subunit of complex III, in two unrelated children presenting with recurrent episodes of ketoacidosis and insulin-responsive hyperglycemia. Cytochrome c₁, the heme-containing component of complex III, mediates the transfer of electrons from the Rieske iron-sulfur protein to cytochrome c. Cytochrome c₁ is present at reduced levels in the skeletal muscle and skin fibroblasts of affected individuals. Moreover, studies on yeast mutants and affected individuals’ fibroblasts have shown that exogenous expression of wild-type CYC1 rescues complex III activity, demonstrating the deleterious effect of each mutation on cytochrome c₁ stability and complex III activity.     

A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples

The recent discovery of 5-hydroxymethyl-cytosine (5hmC) in embryonic stem cells and postmitotic neurons has triggered the need for quantitative measurements of both 5-methyl-cytosine (5mC) and 5hmC in the same sample. We have developed a method using liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (LC–ESI–MS/MS–MRM) to simultaneously measure levels of 5mC and 5hmC in digested genomic DNA. This method is fast, robust, and accurate, and it is more sensitive than the current 5hmC quantitation methods such as end labeling with thin layer chromatography and radiolabeling by glycosylation. Only 50 ng of digested genomic DNA is required to measure the presence of 0.1% 5hmC in DNA from mouse embryonic stem cells. Using this procedure, we show that human induced pluripotent stem cells exhibit a dramatic increase in 5mC and 5hmC levels compared with parental fibroblast cells, suggesting a dynamic regulation of DNA methylation and hydroxymethylation during cellular reprogramming.     

Targeted lymphoid homing of dendritic cells is required for prolongation of allograft survival

Accumulating evidence that dendritic cells (DC) are important regulators of peripheral immune tolerance has led to the concept that donor-derived DC may be useful for inducing donor-specific transplantation tolerance. Although in vitro studies in this field have been encouraging, in vivo results have been inconsistent. Recent evidence has suggested a critical role of lymphoid organs in tolerance induction. In this study, we use a novel gene transduction technique to show that engineered expression of CCR7 on immature DC can markedly increase DC homing to lymphoid organs, leading to increased interaction with Ag-specific T cells. Moreover, we show that a single infusion of DC coexpressing CCR7 and the immunomodulatory molecule viral IL-10 (vIL-10) markedly prolongs cardiac allograft survival (mean survival time >100 days); importantly, DC expressing either vIL-10 alone or CCR7 alone was not effective. These results demonstrate an important paradigm for immune modulation using DC.     

Rhizobium common nod genes are required for biofilm formation

In legume nitrogen-fixing symbioses, rhizobial nod genes are obligatory for initiating infection thread formation and root nodule development. Here we show that the common nod genes, nodD1ABC , whose products synthesize core Nod factor, a chitin-like oligomer, are also required for the establishment of the three-dimensional architecture of the biofilm of Sinorhizobium meliloti . Common nod gene mutants form a biofilm that is a monolayer. Moreover, adding Nod Factor antibody to S. meliloti cells inhibits biofilm formation, while chitinase treatment disrupts pre-formed biofilms. These results attest to the involvement of core Nod factor in rhizobial biofilm establishment. However, luteolin, the plant-derived inducer of S. meliloti 's nod genes, is not required for mature biofilm formation, although biofilm establishment is enhanced in the presence of this flavonoid inducer. Because biofilm formation is plant-inducer-independent and because all nodulating rhizobia, both alpha- and beta-proteobacteria have common nod genes, the role of core Nod factor in biofilm formation is likely to be an ancestral and evolutionarily conserved function of these genes.     

Determination of the composition of the oligosaccharide phosphate fraction of Pichia (Hansenula) holstii NRRL Y-2448 phosphomannan by capillary electrophoresis and HPLC

The promising new anticancer agent, PI-88, is prepared by the sulfonation of the oligosaccharide phosphate fraction of the extracellular phosphomannan produced by the yeast Pichia (Hansenula) holstii NRRL Y-2448. The composition of the oligosaccharide phosphate fraction was determined by capillary electrophoresis (CE) with indirect UV detection using 6 mM potassium sorbate at pH 10.3 as the background electrolyte. Further confirmation of the composition was obtained by HPLC analysis of a sample dephosphorylated by treatment with alkaline phosphatase. The structure of the hexasaccharide component has been determined by isolation and NMR spectroscopic analysis of its dephosphorylated derivative. Additionally, the structure of a second, previously undetected tetrasaccharide component (a hexosamine) has been determined by isolation and NMR spectroscopic analysis of the acetate of its dephosphorylated derivative. It is demonstrated that CE is an ideal method for the quality control of the oligosaccharide phosphate fraction for use in the production of PI-88.     

A 2-Megabase Physical Contig Incorporating 43 DNA Markers on the Human X Chromosome at p11.23–p11.22 from ZNF21 to DXS255

A comprehensive physical contig of yeast artificial chromosomes (YACs) and cosmid clones between ZNF21 and DXS255 has been constructed, spanning 2 Mb within the region Xp11.23–p11.22. As a portion of the region was found to be particularly unstable in yeast, the integrity of the contig is dependent on additional information provided by the sequence-tagged site (STS) content of cosmid clones and DNA marker retention in conventional and radiation hybrids. The contig was formatted with 43 DNA markers, including 19 new STSs from YAC insert ends and an internalAlu-PCR product. The density of STSs across the contig ranges from one marker every 20 kb to one every 60 kb, with an average density of one marker every 50 kb. The relative order of previously known genes and expressed sequence tags in this region is predicted to be Xpter–ZNF21–DXS7465E (MG66)–DXS7927E (MG81)–WASP, DXS1011E, DXS7467E (MG21)–DXS- 7466E (MG44)–GATA1–DXS7469E (Xp664)–TFE3–SYP (DXS1007E)–Xcen. This contig extends the coverage in Xp11 and provides a framework for the future identification and mapping of new genes, as well as the resources for developing DNA sequencing templates.