High Expression of the HNK-1/L2 Carbohydrate Epitope in the Major Glycoproteins of Shark Myelin

The major 24- and 28-kDa glycoproteins in shark PNS and CNS myelin express high levels of the adhesion-associated HNK-1/L2 carbohydrate epitope. The 28-kDa protein, but not the 24-kDa protein, cross-reacts strongly with one of two anti-bovine P0 antisera not previously tested against fish myelin proteins. Shark PNS and CNS myelin also contains smaller amounts of high-molecular-weight HNK-1-positive proteins, including a prominent broad band in the 65-85-kDa range. Although myelin-associated glycoprotein (MAG) is well known to react with HNK-1 in some mammals, monoclonal and polyclonal anti-MAG antibodies did not react with the high-molecular-weight HNK-1-positive material in shark myelin, a result suggesting that it is not a MAG-like protein. The high expression of the HNK-1/L2 epitope in glycoproteins of shark myelin, including the major P0-related ones, suggests that this adhesion-related carbohydrate structure may have had an important role in the molecular evolution of the myelinating process.     

Susceptibility of the Myelin-Associated Glycoprotein and Basic Protein to a Neutral Protease in Highly Purified Myelin from Human and Rat Brain

Abstract: Incubation of highly purified human myelin at 25° and pH 8 in ammonium bicarbonate buffer resulted in the conversion of the myelin-associated glycoprotein (MAG) to a smaller derivative (dMAG) with an apparent molecular weight about 10,000 less. dMAG was stable and was not degraded to lower-molecular-weight breakdown products. Incubation of myelin under these conditions also resulted in the degradation of basic protein, but at a much slower rate. Half of the MAG was converted to dMAG in about 30 min, whereas degradation of half of the basic protein required 18 h of incubation. There was no significant loss of proteolipid, the Wolfgram doublet, or other myelin proteins during incubation for up to 18 h under these conditions. The formation of dMAG and the degradation of basic protein appear to be mediated by similar enzymatic activities; both processes exhibited broad pH optima in the neutral range, were prevented by briefly heating the myelin to 70° before incubation, and were stimulated by ammonium bicarbonate and other salts. Incubation of purified rat myelin also resulted in the formation of dMAG and the degradation of basic protein, but the conversion to dMAG occurred much more slowly than in human myelin preparations. In the rat, the percentage decreases in intact MAG and in basic protein were similar to each other and proceeded at rates comparable to the loss of basic protein in human myelin. These studies confirm and extend earlier demonstrations of neutral protease activity in purified myelin, and show that cleavage of MAG is one of the effects of this activity. The proteolytic activity affecting MAG and basic protein was not significantly reduced by further purification of the myelin on sucrose or CsCl gradients, suggesting that the neutral protease may be a myelin-related enzyme. The very high susceptibility of human MAG to this enzyme indicates that the effect of neutral protease on this glycoprotein should be considered in connection with demyelinating diseases.     

Conditional Deletion of Fgfr1 in the Proximal and Distal Tubule Identifies Distinct Roles in Phosphate and Calcium Transport

A postnatal role of fibroblast growth factor receptor-1 (FGFR1) in the kidney is suggested by its binding to α-Klotho to form an obligate receptor for the hormone fibroblast growth factor-23 (FGF-23). FGFR1 is expressed in both the proximal and distal renal tubular segments, but its tubular specific functions are unclear. In this study, we crossed Fgfr1^flox/flox mice with either gamma-glutamyltransferase-Cre (γGT-Cre) or kidney specific-Cre (Ksp-Cre) mice to selectively create proximal tubule (PT) and distal tubule (DT) Fgfr1 conditional knockout mice (designated Fgfr1^PT-cKOand Fgfr1^DT-cKO, respectively). Fgfr1^PT-cKO mice exhibited an increase in sodium-dependent phosphate co-transporter expression, hyperphosphatemia, and refractoriness to the phosphaturic actions of FGF-23, consistent with a direct role of FGFR1 in mediating the proximal tubular phosphate responses to FGF-23. In contrast, Fgfr1^DT-cKO mice unexpectedly developed hypercalciuria, secondary elevations of parathyroid hormone (PTH), hypophosphatemia and enhanced urinary phosphate excretion. Fgfr1^PT-cKO mice also developed a curly tail/spina bifida-like skeletal phenotype, whereas Fgfr1^DT-cKO mice developed renal tubular micro-calcifications and reductions in cortical bone thickness. Thus, FGFR1 has dual functions to directly regulate proximal and distal tubule phosphate and calcium reabsorption, indicating a physiological role of FGFR1 signaling in both phosphate and calcium homeostasis.     

Mapping ubiquitination sites of S. cerevisiae Mcm10

Minichromosome maintenance protein (Mcm) 10 is a part of the eukaryotic replication machinery and highly conserved throughout evolution. As a multivalent DNA scaffold, Mcm10 coordinates the action of proteins that are indispensable for lagging strand synthesis, such as the replication clamp, proliferating cell nuclear antigen (PCNA). The binding between Mcm10 and PCNA serves an essential function during DNA elongation and is mediated by the ubiquitination of Mcm10. Here we map lysine 372 as the primary attachment site for ubiquitin on S. cerevisiae Mcm10. Moreover, we identify five additional lysines that can be ubiquitinated. Mutation of lysine 372 to arginine ablates ubiquitination of overexpressed protein and causes sensitivity to the replication inhibitor hydroxyurea in cells that are S-phase checkpoint compromised. Together, these findings reveal the high selectivity of the ubiquitination machinery that targets Mcm10 and that ubiquitination has a role in suppressing replication stress.     

[3 5S]Sulfate incorporation into myelin glycoproteins II. Peripheral nervous tissue

The in vivo incorporation of [^{3 5}S]sulfate, [³H]fucose and [³H]leucine into sciatic nerve myelin was investigated. Polyacrylamide gel electrophoresis of the proteins indicated that the ^{3 5}S-labeling of proteins occurred almost exclusively in the major myelin protein. A smaller myelin glycoprotein migrating just ahead of the major one was labeled with [³H]fucose but did not incorporate ^{3 5}S to a detectable extent. There was little or no ^{3 5}S associated with basic proteins on polyacrylamide gels when the proteins were extracted with chloroform/methanol. Fucose-labeled myelin glycoproteins were converted to glycopeptides by pronase digestion. The glycopeptides gave a single peak on Sephadex G-50 in which the ³H and ^{3 5}S coincided. The association of ^{3 5}S with glycopeptides was not caused by binding of sulfatide or free inorganic sulfate. This study shows that the major myelin protein in the sciatic nerve of the rat is glycosylated and sulfated.     

High density DNA methylation array with single CpG site resolution

We have developed a new generation of genome-wide DNA methylation BeadChip which allows high-throughput methylation profiling of the human genome. The new high density BeadChip can assay over 480K CpG sites and analyze twelve samples in parallel. The innovative content includes coverage of 99% of RefSeq genes with multiple probes per gene, 96% of CpG islands from the UCSC database, CpG island shores and additional content selected from whole-genome bisulfite sequencing data and input from DNA methylation experts. The well-characterized Infinium® Assay is used for analysis of CpG methylation using bisulfite-converted genomic DNA. We applied this technology to analyze DNA methylation in normal and tumor DNA samples and compared results with whole-genome bisulfite sequencing (WGBS) data obtained for the same samples. Highly comparable DNA methylation profiles were generated by the array and sequencing methods (average R² of 0.95). The ability to determine genome-wide methylation patterns will rapidly advance methylation research.     

Spatially resolved characterization of biogenic manganese oxide production within a bacterial biofilm

Pseudomonas putida strain MnB1, a biofilm-forming bacterial culture, was used as a model for the study of bacterial Mn oxidation in freshwater and soil environments. The oxidation of aqueous Mn+2 [Mn+2(aq)] by P. putida was characterized by spatially and temporally resolving the oxidation state of Mn in the presence of a bacterial biofilm, using scanning transmission X-ray microscopy (STXM) combined with near-edge X-ray absorption fine structure (NEXAFS) spectroscopy at the Mn L2,3 absorption edges. Subsamples were collected from growth flasks containing 0.1 and 1 mM total Mn at 16, 24, 36, and 48 h after inoculation. Immediately after collection, the unprocessed hydrated subsamples were imaged at a 40-nm resolution. Manganese NEXAFS spectra were extracted from X-ray energy sequences of STXM images (stacks) and fit with linear combinations of well-characterized reference spectra to obtain quantitative relative abundances of Mn(II), Mn(III), and Mn(IV). Careful consideration was given to uncertainty in the normalization of the reference spectra, choice of reference compounds, and chemical changes due to radiation damage. The STXM results confirm that Mn+2(aq) was removed from solution by P. putida and was concentrated as Mn(III) and Mn(IV) immediately adjacent to the bacterial cells. The Mn precipitates were completely enveloped by bacterial biofilm material. The distribution of Mn oxidation states was spatially heterogeneous within and between the clusters of bacterial cells. Scanning transmission X-ray microscopy is a promising tool for advancing the study of hydrated interfaces between minerals and bacteria, particularly in cases where the structure of bacterial biofilms needs to be maintained.     

Changing Museum Visitors’ Conceptions of Evolution

We examined whether a single visit to an evolution exhibition contributed to conceptual change in adult (n?=?30), youth, and child (n?=?34) museum visitors?? reasoning about evolution. The exhibition included seven current research projects in evolutionary science, each focused on a different organism. To frame this study, we integrated a developmental model of visitors?? understanding of evolution, which incorporates visitors?? intuitive beliefs, with a model of free-choice learning that includes personal, sociocultural, and contextual variables. Using pre- and post-measures, we assessed how visitors?? causal explanations about biological change, drawn from three reasoning patterns (evolutionary, intuitive, and creationist), were modified as a result of visiting the exhibition. Whatever their age, background beliefs, or prior intuitive reasoning patterns, visitors significantly increased their use of explanations from the evolutionary reasoning pattern across all measures and extended this reasoning across diverse organisms. Visitors also increased their use of one intuitive reasoning pattern, need-based (goal-directed) explanations, which, we argue, may be a step toward evolutionary reasoning. Nonetheless, visitors continued to use mixed reasoning (endorsing all three reasoning patterns) in explaining biological change. The personal, socio-cultural, and contextual variables were found to be related to these reasoning patterns in predictable ways. These findings are used to examine the structure of visitors?? reasoning patterns and those aspects of the exhibition that may have contributed to the gains in museum visitors?? understanding of evolution.     

Effects of Congenital Infection of Sheep with Border Disease Virus on Myelin Proteins

Abstract— Border disease (BD) of sheep is caused by a virus in the genus Pestivirus that results in decreased myelination throughout the CMS when acquired congenitally. Pregnant ewes were inoculated with BD virus at 50 days of gestation, and myelin proteins were quantified in several regions of the CNS during prenatal and postnatal development of infected lambs for comparison with age-matched controls. Newborn field-infected lambs were also examined. Myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) were measured by densitometric scanning of western blots. Deficiencies in the myelin proteins were detected as early as 116 days of gestation, and the deficiencies of myelin proteins were most pronounced in the cerebellum at all ages examined. PLP and MBP increased from 10–30% of normal in cerebellar white matter at birth to 40–60% of normal at 6 months, suggesting some catch-up in the amount of compact myelin with development. MAG and CNP were between 70 and 80% of control levels in the cerebellum at birth and at 6 months. Similar results were obtained for the corpus callosum and spinal cord of infected lambs, but the deficiencies of myelin proteins were not as great. A common finding in all regions examined was that MBP and PLP were reduced more than MAG and CNP. This is probably explained by a greater deficit of compact myelin, in which MBP and PLP are localized, than of associated oli-godendroglial membranes, in which MAG and CNP are concentrated. Similar results have been obtained in several dysmyelinating mutants, pointing to common factors in virally and genetically caused hypomyelination. Key Words: Border disease—Myelin—Hypomyelination—Development—Sheeo.