Dystrophin is a microtubule-associated protein

Cytolinkers are giant proteins that can stabilize cells by linking actin filaments, intermediate filaments, and microtubules (MTs) to transmembrane complexes. Dystrophin is functionally similar to cytolinkers, as it links the multiple components of the cellular cytoskeleton to the transmembrane dystroglycan complex. Although no direct link between dystrophin and MTs has been documented, costamere-associated MTs are disrupted when dystrophin is absent. Using tissue-based cosedimentation assays on mice expressing endogenous dystrophin or truncated transgene products, we find that constructs harboring spectrinlike repeat 24 through the first third of the WW domain cosediment with MTs. Purified Dp260, a truncated isoform of dystrophin, bound MTs with a K_d of 0.66 µM, a stoichiometry of 1 Dp260/1.4 tubulin heterodimer at saturation, and stabilizes MTs from cold-induced depolymerization. Finally, α- and β-tubulin expression is increased ∼2.5-fold in mdx skeletal muscle without altering the tubulin–MT equilibrium. Collectively, these data suggest dystrophin directly organizes and/or stabilizes costameric MTs and classifies dystrophin as a cytolinker in skeletal muscle.     

PDA: Pooled DNA analyzer

Background  

Association mapping using abundant single nucleotide polymorphisms is a powerful tool for identifying disease susceptibility genes for complex traits and exploring possible genetic diversity. Genotyping large numbers of SNPs individually is performed routinely but is cost prohibitive for large-scale genetic studies. DNA pooling is a reliable and cost-saving alternative genotyping method. However, no software has been developed for complete pooled-DNA analyses, including data standardization, allele frequency estimation, and single/multipoint DNA pooling association tests. This motivated the development of the software, 'PDA' (Pooled DNA Analyzer), to analyze pooled DNA data.     

Transmembrane partitioning of boron and other elements in RAW 264.7 and HL60 cell cultures

The trace element boron is essential for all higher plants and is beneficial or has been established as essential for several animal models of human nutrition. To help identify the biomolecules that require boron for function in humans, we determined whether intracellular boron is retained against a concentration gradient. Cells (Abelson leukemia virus BALB murine monocyte-macrophage RAW 264.7 [RAW] and HL60) and supplemented media (Dulbecco’s modified essential media [+10% fetal calf serum] and Iscove’s modified Dulbecco’s medium [+5% fetal calf serum], respectively) were analyzed for mineral concentrations after culture and subculture. Special corrections were made for trapped extracellular media in cell pellets and endocytosed media. For RAW cells, the partitioning coefficients (PC; intracellular/extracellular ratios) were, in rank order, as follows: Mn, 110; Fe, 67; P, 65; Zn, 32; K, 15; Cu, 7.1; Mg, 4.3; B, 1.7; Ca, 0.4; Na, 0.3. For HL60 cells, the partitioning coefficients were, in rank order, as follows: Mn, 212; Zn, 211; P, 123; K, 21; Fe, 16; Mg, 11; B, 1.7; Ca, 0.8; Na, 0.3. Trapped extracellular media was estimated to be 6.7±0.8%; trapped extracellular and endocytosed media together was 24.8±0.3% of the mass within the isolated cell pellets. The partitioning coefficients indicate a positive gradient for intracellular accumulation of boron, zinc, phosphorus, managanese, magnesium, potassium, iron, and copper in RAW264.7 and HL60 cells. Specifically, the data indicate the existence of a selective boron-binding molecular species within the cell or the existence of a boron-specific membrane transporter.     

Exploring the whereabouts of GLUT4 in skeletal muscle (review)

The glucose transporter GLUT4 is expressed in muscle, fat cells, brain and kidney. In contrast to other glucose transporters, GLUT4 in unstimulated cells is mostly intracellular. Stimuli such as insulin and muscle contractions then cause the translocation of GLUT4 to the cell surface. Questions related to GLUT4 storage compartments, trafficking to the surface membrane, and nature of the intracellular pools, have kept many groups busy for the past 20 years. Yet, one of the main questions in the field remains the universality of GLUT4 features. Can one extrapolate work done on fat cells to muscle or brain? Or vice-versa? Can one use cultures to predict GLUT4 behaviour in fully differentiated tissues? This review summarizes the authors' knowledge of GLUT4 biology in skeletal muscle, which is the predominant tissue for glucose homeostasis. The results are compared to those obtained with the fat cell system, and an attempt is made to assess the universality principle.     

Linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of the Tetrahymena ribozyme

We have explored the linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of Tetrahymena thermophila ribozyme by examining the Mg2+-induced folding and the urea-induced denaturation of the folded state as a function of Na+ under equilibrium folding conditions using hydroxyl radical footprinting. These studies allowed a thermodynamic examination of eight discrete protection sites within P4-P6 that are involved in several tertiary structure contacts. Monovalent ions compete with Mg2+ ions in mediating P4-P6 folding. The urea denaturation isotherms demonstrated DeltaDeltaG values of >2 kcal x mol(-1) in experiments conducted in 10 versus 200 mM NaCl at a constant 10 mM MgCl2. However, the individual-site isotherms reported by footprinting revealed that larger than average changes in DeltaG values were localized to specific sites within the Mg2+-rich A-bulge. The competitive effects of monovalent ions were less when K+ rather than Na+ was the monovalent cation present. This result indicates the importance of the specific K+ binding sites that are associated with AA-platform structures to P4-P6 folding and stability. These site-specific footprinting data provide quantitative and site-specific measurements of the ion-linked stability for P4-P6 that are interpreted with respect to crystallographic data.     

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.