Formalin removal from archival tissue by critical point drying

The extraction of high-quality nucleic acid may be problematic in formalin-fixed tissues because of cross-linking between proteins and DNA. Old fixed tissue specimens do produce fragmented DNA (<1.2 kb), which is only used for PCR amplification. Here we show that high molecular weight DNA (>194 kb) can be successfully extracted from fixed tissue samples (16-70 years old) by gradual dehydration and critical point drying. The reliability of extracted DNA was measured by its ability to serve as a template for the amplification of mtDNA fragments (403 and 1198 bp) and an nDNA fragment (1844 bp). In addition, fingerprinting analysis was performed using DNA from fixed human tissue to ensure the ability of extracted DNA to hybridize with the DNA probe. DNA derived by this method can be subject to amplification, complete digestion by restriction endonuclease, and hybridization.     

Thermal induced modification of the contact mechanics of adhering liposomes on cationic substrate

The correlation between the mechanical property and the thermotropic transition of the phospholipid bilayer has been recently demonstrated (Chem. Phys. Lipids 110 (2001) 27). However, the role of thermal induced mechanical responses of phospholipid bilayer on the contact mechanics of liposome adhering on a cationic substrate has not been determined. In this study, confocal-reflectance interference contrast microscopy, phase contrast microscopy and contact mechanics modeling are applied to probe the adhesion mechanisms of liposomes in the presence of electrostatic interactions during the thermotropic transition of the lipid bilayer. When temperature increases from 23 to 49 °C at pH 7.4, the degree of liposome deformation (a/R) and adhesion energy of dipalmitoyl-sn-glycero-3-phosphocholine liposome increases by 10% and remains constant, respectively, on 3-amino-propyl-triethoxy-silane (APTES) modified substrate. The extents of increase in these two parameters are highly dependent on the physicochemical properties of the rigid substrate. At pH 4, the adhesion energies above and below the phase transition temperature (T_m) are increased by one order of magnitude due to the formation of the free silanol groups on APTES substrate. In hypotonic condition, the degree of vesicle deformation remains constant and the adhesion energy reduces by 20% during sample heating. Under all conditions, the adhesion energy of the adhering liposome spans a few orders of magnitude against the increase of liposome size as the surface area to volume ratio is maximized in smallest vesicle.     

Twist function is required for the morphogenesis of the cephalic neural tube and the differentiation of the cranial neural crest cells in the mouse embryo

Loss of Twist function in the cranial mesenchyme of the mouse embryo causes failure of closure of the cephalic neural tube and malformation of the branchial arches. In the Twist(-/-) embryo, the expression of molecular markers that signify dorsal forebrain tissues is either absent or reduced, but those associated with ventral tissues display expanded domains of expression. Dorsoventral organization of the mid- and hindbrain and the anterior-posterior pattern of the neural tube are not affected. In the Twist(-/-) embryo, neural crest cells stray from the subectodermal migratory path and the late-migrating subpopulation invades the cell-free zone separating streams of cells going to the first and second branchial arches. Cell transplantation studies reveal that Twist activity is required in the cranial mesenchyme for directing the migration of the neural crest cells, as well as in the neural crest cells within the first branchial arch to achieve correct localization. Twist is also required for the proper differentiation of the first arch tissues into bone, muscle, and teeth.     

Regulation of TRKB surface expression by brain-derived neurotrophic factor and truncated TRKB isoforms

Brain-derived neurotrophic factor (BDNF) signaling through its receptor TRKB modulates survival, differentiation, and activity of neurons. BDNF activates TRKB on the cell surface, which leads to the initiation of intracellular signaling cascades and different biological responses in neurons. Neuronal activity has been shown to regulate TRKB levels on the plasma membrane of neurons, but little is known about other factors affecting TRKB surface expression levels. We report here that BDNF regulates the cell surface levels of transfected or endogenously expressed full-length TRKB, depending on the exposure time in neuroblastoma cells and primary hippocampal neurons. BDNF rapidly increases TRKB surface expression levels in seconds, whereas treatment of cells with BDNF for a longer time (minutes to hours) leads to decreased TRKB surface levels. Coexpression of the full-length TRKB together with the truncated TRKB.T1 isoform results in decreased levels of full-length TRKB on the cell surface. This effect is specific to the T1 isoform, because coexpression of a kinase-dead TRKB mutant or another kinase domain-lacking TRKB form, truncated T-Shc, leads to increased TRKB surface levels. Our results suggest that regulation of TRKB surface expression levels by different factors is tightly controlled by complex mechanisms in active neurons.     

Erythroid expression of the human alpha-spectrin gene promoter is mediated by GATA-1- and NF-E2-binding proteins

alpha-Spectrin is a highly expressed membrane protein critical for the flexibility and stability of the erythrocyte. Qualitative and quantitative defects of alpha-spectrin are present in the erythrocytes of many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. We wished to determine the regulatory elements that determine the erythroid-specific expression of the alpha-spectrin gene. We mapped the 5' end of the alpha-spectrin erythroid cDNA and cloned the 5' flanking genomic DNA containing the putative alpha-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, an alpha-spectrin gene erythroid promoter with binding sites for GATA-1- and NF-E2-related proteins was identified. Both binding sites were required for full promoter activity. In transgenic mice, a reporter gene directed by the alpha-spectrin promoter was expressed in yolk sac, fetal liver, and erythroid cells of bone marrow but not adult reticulocytes. No expression of the reporter gene was detected in nonerythroid tissues. We conclude that this alpha-spectrin gene promoter contains the sequences necessary for low level expression in erythroid progenitor cells.     

The soft metal ion binding sites in the Staphylococcus aureus pI258 CadC Cd(II)/Pb(II)/Zn(II)-responsive repressor are formed between subunits of the homodimer

The Staphylococcus aureus plasmid pI258 CadC is a homodimeric repressor that binds Cd(II), Pb(II), and Zn(II) and regulates expression of the cadAC operon. CadC binds two Cd(II) ions per dimer, with a tetrathiolate binding site composed of residues Cys(7), Cys(11), Cys(58), and Cys(60). It is not known whether each site consists of residues from a single monomer or from residues contributed by both subunits. To examine whether Cys(7) and Cys(11) are spatially proximate to Cys(58) and Cys(60) of the same subunit or of the other subunit, homodimers with the same cysteine mutation in each subunit and heterodimers containing different cysteine mutations in the two subunits were reacted with 4,6-bis(bromomethyl)-3,7-dimethyl-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione, which cross-links thiol groups that are within 3-6 A of each other. Cys(7) or Cys(11) cross-linked only with Cys(58) or Cys(60) on the other subunit. The data demonstrate that Cys(7) and Cys(11) from one monomer are within 3-6 A of either Cys(58) or Cys(60) in the other monomer. The results of this study strongly indicate that each of the two Cd(II) binding sites in the CadC homodimer is composed of Cys(7) and Cys(11) from one monomer and Cys(58) and Cys(60) from the other monomer.     

Conserved and nonconserved features of the folding pathway of hisactophilin, a beta-trefoil protein

Based on previous studies of interleukin-1beta (IL-1beta) and both acidic and basic fibroblast growth factors (FGFs), it has been suggested that the folding of beta-trefoil proteins is intrinsically slow and may occur via the formation of essential intermediates. Using optical and NMR-detected quenched-flow hydrogen/deuterium exchange methods, we have measured the folding kinetics of hisactophilin, another beta-trefoil protein that has < 10% sequence identity and unrelated function to IL-1beta and FGFs. We find that hisactophilin can fold rapidly and with apparently two-state kinetics, except under the most stabilizing conditions investigated where there is evidence for formation of a folding intermediate. The hisactophilin intermediate has significant structural similarities to the IL-1beta intermediate that has been observed experimentally and predicted theoretically using a simple, topology-based folding model; however, it appears to be different from the folding intermediate observed experimentally for acidic FGF. For hisactophilin and acidic FGF, intermediates are much less prominent during folding than for IL-1beta. Considering the structures of the different beta-trefoil proteins, it appears that differences in nonconserved loops and hydrophobic interactions may play an important role in differential stabilization of the intermediates for these proteins.