A Polyadenylation Factor Subunit Implicated in Regulating Oxidative Signaling in Arabidopsis thaliana

BACKGROUND: Plants respond to many unfavorable environmental conditions via signaling mediated by altered levels of various reactive oxygen species (ROS). To gain additional insight into oxidative signaling responses, Arabidopsis mutants that exhibited tolerance to oxidative stress were isolated. We describe herein the isolation and characterization of one such mutant, oxt6. METHODOLOGY/PRINCIPAL FINDINGS: The oxt6 mutation is due to the disruption of a complex gene (At1g30460) that encodes the Arabidopsis ortholog of the 30-kD subunit of the cleavage and polyadenylation specificity factor (CPSF30) as well as a larger, related 65-kD protein. Expression of mRNAs encoding Arabidopsis CPSF30 alone was able to restore wild-type growth and stress susceptibility to the oxt6 mutant. Transcriptional profiling and single gene expression studies show elevated constitutive expression of a subset of genes that encode proteins containing thioredoxin- and glutaredoxin-related domains in the oxt6 mutant, suggesting that stress can be ameliorated by these gene classes. Bulk poly(A) tail length was not seemingly affected in the oxt6 mutant, but poly(A) site selection was different, indicating a subtle effect on polyadenylation in the mutant. CONCLUSIONS/SIGNIFICANCE: These results implicate the Arabidopsis CPSF30 protein in the posttranscriptional control of the responses of plants to stress, and in particular to the expression of a set of genes that suffices to confer tolerance to oxidative stress

Stereodynamics and characterization of the hexa(4-n-dodecylbiphenylyl)benzene hexaanion that includes a twisted benzene core

Hexa(4-n-dodecylbiphenylyl)benzene (HDBB) was reduced by a series of alkali metals in THF under high vacuum. Three reduction states were identified by NMR spectroscopy, namely the dianion, tetraanion and hexaanion. The NMR spectra of HDBB^6- revealed a remarkable distortion of symmetry, which is interpreted by adoption of a twisted conformation of the central benzene ring and a slow rotation of the inner phenylene rings of the biphenyl units. Due to the surprising thermal stability of the hexaanion, a dynamic NMR investigation revealed the pseudorotation of the twisted conformation and the phenylene rotation mentioned above

Stereodynamics and characterization of the hexa(4-n-dodecylbiphenylyl)benzene hexaanion that includes a twisted benzene core

Hexa(4-n-dodecylbiphenylyl)benzene (HDBB) was reduced by a series of alkali metals in THF under high vacuum. Three reduction states were identified by NMR spectroscopy, namely the dianion, tetraanion and hexaanion. The NMR spectra of HDBB6- revealed a remarkable distortion of symmetry, which is interpreted by adoption of a twisted conformation of the central benzene ring and a slow rotation of the inner phenylene rings of the biphenyl units. Due to the surprising thermal stability of the hexaanion, a dynamic NMR investigation revealed the pseudorotation of the twisted conformation and the phenylene rotation mentioned above

Purification and identification of two putative autolytic sites in human calpain 3 (p94) expressed in heterologous systems

Human muscle-specific calpain (CAPN3) was expressed in two heterologous systems: Sf9 insect cells and Escherichia coli cells. Polyclonal antibodies were prepared against peptides whose sequences were taken from the three unique regions of human CAPN3, namely NS, IS1, and IS2, which are not found in other members of the calpain family. Western blot analysis using these antibodies revealed that CAPN3 was well expressed in both systems. However, considerable rapid degradation of the expressed CAPN3 was observed in both Sf9 and E. coli cells. These antibodies were therefore also used to detect CAPN3 and its degradation products in human and rat muscles, as well as to detect the protein throughout the purification of the recombinant His-tagged human CAPN3 by Ni2+ affinity chromatography and by immunopurification over immobilized antibody. An alternative purification procedure was used for purification of all putative CAPN3 immunoreactive fragments by combining SDS-PAGE and hydroxyapatite chromatography. Two fragments of CAPN3 of approximately 55 kDa were purified, and their N-terminal amino acid sequencing demonstrated that cleavage of CANP3 occurred between residues 30-31 and 412-413, thus providing the first evidence for the localization of putative autolytic sites in this enzyme