A Note on the Local Current Associated with the Rising Phase of a Propagating Impulse in Nonmyelinated Nerve Fibers

To extend our recent paper dealing with the cable properties and the conduction velocity of nonmyelinated nerve fibers (Bull. Math. Biol. 64, 1069; 2002), the behavior of the local current associated with the rising phase of a propagating action potential is discussed. It is shown that the process of charging the membrane capacity by means of the local current plays a crucial role in determining the velocity of nerve conduction. The symmetry of the local current with respect to the boundary between the resting and active regions of the nerve fiber is emphasized. It is noted that there are several simple quantitative rules governing the intensities of the capacitive, resistive and total membrane currents observed during the rising phase of an action potential.     

Microplate assay for aptamer-based thrombin detection using a DNA-enzyme conjugate based on histidine-tag chemistry

We report a method to prepare a DNA–enzyme conjugate using histidine-tag (His-tag) chemistry. A DNA oligonucleotide was modified with nitrilotriacetate (NTA), whose K_d was approximately 10^?6 (M^?1) toward a His-tag present on a recombinant protein via the complexation of Ni²⁺. His-tagged alkaline phosphatase (His-AP) was used as the model enzyme. Enzyme immobilization on the microplate revealed the conjugation of His-AP and the NTA-modified DNA via an Ni²⁺ complex. SPR measurements also proved the conjugation of His-AP with the NTA-modified DNA via an Ni²⁺ complex. The DNA–enzyme conjugate was then used for the detection of thrombin using a DNA aptamer. The DNA-AP conjugate successfully amplified the binding signal between the DNA aptamer and the thrombin, and the signal was measured as the fluorescent intensity derived from the AP-catalyzed reaction. The detection limit was 11 nM. Finally, we studied the effect of the release of the immobilized His-AP from the microplate on the AP activity, because the present strategy used a cleavable linker for the conjugation and the enzyme immobilization. The DNase-catalyzed release of the immobilized His-AP resulted in a 1.7-fold higher AP activity than observed when the His-AP was surface-immobilized.     

Identification and characterization of xanthone biosynthetic genes contributing to the vivid red coloration of red-flowered gentian

Cultivated Japanese gentians traditionally produce vivid blue flowers because of the accumulation of delphinidin-based polyacylated anthocyanins. However, recent breeding programs developed several red-flowered cultivars, but the underlying mechanism for this red coloration was unknown. Thus, we characterized the pigments responsible for the red coloration in these cultivars. A high-performance liquid chromatography with photodiode array analysis revealed the presence of phenolic compounds, including flavones and xanthones, as well as the accumulation of colored cyanidin-based anthocyanins. The chemical structures of two xanthone compounds contributing to the coloration of red-flowered gentian petals were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds were identified as norathyriol 6-O-glucoside (i.e., tripteroside designated as Xt1) and a previously unreported norathyriol-6-O-(6′-O-malonyl)-glucoside (designated Xt2). The copigmentation effects of these compounds on cyanidin 3-O-glucoside were detected in vitro. Additionally, an RNA sequencing analysis was performed to identify the cDNAs encoding the enzymes involved in the biosynthesis of these xanthones. Recombinant proteins encoded by the candidate genes were produced in a wheat germ cell-free protein expression system and assayed. We determined that a UDP-glucose-dependent glucosyltransferase (StrGT9) catalyzes the transfer of a glucose moiety to norathyriol, a xanthone aglycone, to produce Xt1, which is converted to Xt2 by a malonyltransferase (StrAT2). An analysis of the progeny lines suggested that the accumulation of Xt2 contributes to the vivid red coloration of gentian flowers. Our data indicate that StrGT9 and StrAT2 help mediate xanthone biosynthesis and contribute to the coloration of red-flowered gentians via copigmentation effects.     

The Murine Th2 Locus Undergoes Epigenetic Modification in the Thymus during Fetal and Postnatal Ontogeny

Epigenetic modifications play a central role in the differentiation and function of immune cells in adult animals. Developmentally regulated epigenetic patterns also appear to contribute to the ontogeny of the immune system. We show here that the epigenetic profile of the T-helper (Th) 2 locus undergoes changes in T lineage cells beginning in mid-gestation and extending throughout the first week of life. In particular, regulatory regions of the Th2 locus are largely methylated at CpG residues among fetal liver common lymphoid progenitor cells. The locus subsequently becomes highly hypomethylated among the downstream progeny of these cells within the fetal thymus. This hypomethylated state is preserved until birth when the locus becomes rapidly re-methylated, achieving adult-like status by 3–6 days post birth. Notably, the capacity for rapid, high level Th2 cytokine production is lost in parallel with this re-methylation. In vitro organ culture and in vivo transplantation experiments indicate that signals from the adult environment are required to achieve the postnatal methylated state. Together, these findings indicate that the Th2 bias of neonates may be conferred, in part, by an epigenetic profile inherited from fetal life. However, the fetal program is rapidly terminated post birth by the development of signals leading to the acquisition of adult-like epigenetic patterns.     

Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA

Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration.     

UV Photoelectron Spectroscopy and Ab Initio Characterization of Valence Orbital Structures and Conformations of Neutral Phosphate Esters

Abstract

The Hel UV photoelectron spectrum of trimethyl phosphate (TMP) has been measured and interpreted with the aid of SCF molecular orbital calculations carried out with STO-3G, STO-3G* and 4–31G basis functions. The photoelectron spectrum of TMP is more accurately reproduced by results from 4–31G calculations than by results from STO-3G or STO-3G* calculations. However, all three basis sets yield results which predict the same assignment of the photoelectron spectrum. Results at the 4–31G level indicate that whether calculations are based on crystallographic bond angles and bond lengths or on STO-3G optimized geometries has little effect on the energetic ordering of the upper occupied orbitals. The energetic ordering of orbitals is also found to be only weakly dependent upon the torsional angle φ, describing rotation of ester groups about P-O bonds and upon the torsional angle ψ, describing rotation of methyl groups about C-O bonds. For trimethyl phosphate, with C₃ symmetry, the vertical ionization potentials of the upper occupied orbitals are 10.81 eV (8e), 11.4 eV (9a), 11.93 eV (7e), 12.6–12.9 eV (8a and 6e), 14.4 eV (7a) and 15.0–16.0 eV(5e and 6a). Calculations at the 4–31G level indicate that many of the highest occupied orbitals in neutral dimethyl phosphate and methyl phosphate have energies and electron distributions similar to orbitals in TMP.

For TMP, a search for optimized values of φ and ψ has been carried out at the STO-3G* level. In agreement with previous NMR studies and with classical potential calculations, the STO- 3G* results indicate that both the gauche φ= 53.1 °) and anticlinal (φ = 141.9°) conformations are thermally accessible. Also in agreement with the classical potential calculations, the STO-3G* results predict that in the all gauche conformation energy is minimized when the methyl groups assume a staggered geometry (ψ= 60° to 80°) and that an energy maximum occurs for an eclipsed geometry (ψ = 0° to 20°). A study of the dependence of optimized values of O-P-O ester bond angles on the torsional angles, φ, was carried out at the STO-3G, STO-3G* and 4–31G levels. The results demonstrate that for C₃ symmetry, the coupling of O-P-O angles to φ is influenced by repulsive steric interactions.