Insect odorant receptors: channeling scent

Odorant detection in insects involves heterodimers between an odorant receptor (OR) and a conserved seven-transmembrane protein called Or83b, but the exact mechanism of OR signal transduction is unclear. Two recent studies in Nature (Sato et al., 2008; Wicher et al., 2008) now reveal that these OR-Or83b heterodimers form odorant-gated ion channels, revealing a surprising new mode of olfactory transduction.     

Fatty acid composition of total lipids from the needles of pine seedlings as related to boron availability

The effect of boron availability on the content of fatty acids (FA) in the total lipids of needles was investigated in two-year-old seedlings of Scots pine (Pinus sylvestris L.). The study was conducted in the forest nursery on the soil deficient in boron. Various rates of boric acid (0.01, 0.03, 0.1, 0.3, 1, 3, and 10 g/m²) were applied to soil three times throughout the growth period. When pine seedlings were supplied with extra boron, the lipid content of the seedling needles declined. The optimal boron supply elevated the content of saturated FA in total needle lipids mostly at the expense of palmitic acid, with a corresponding decline in the level of unsaturated FA and unsaturation index of FA owing to trienoic FA (mainly linolenic and hexadecatrienoic acids). At the same time, the level of monoenoic and to a lesser degree dienoic acids increased. When boron supply of the seedlings was not optimal, the content of unsaturated and low-molecular FA increased.     

Identification and gene expression profiling of the Pum1 and Pum2 members of the Pumilio family in the chicken

Members of the Pumilio (Pum) family of RNA-binding proteins act as translational repressors and are required for germ cell development and asymmetric division. We identified the chicken Pum1 and Pum2 genes and analyzed their expression patterns in various tissues. Comparative sequence analysis of the Pum1 and Pum2 proteins from the drosophila, chicken, mouse, and human revealed a high degree of evolutionary conservation in terms of the levels of homology of the peptide sequences and the structure of Pumilio homology domain (PUM-HD), C-terminal RNA-binding domain, with similar spacing between the adjacent Pum eight tandem repeats. In addition, phylogenetic patterns of pumilio family showed that Pum 1 and 2 of chicken are more closely related to those of mouse and human than other species and Pum1 is more conserved than Pum2. Using real-time RT-PCR, the expression levels of the Pum1 and Pum2 genes were found to be highest in hatched female gonads, and high-level expression of Pum2 was detected in 12-day and hatched gonads among the various chicken embryonic tissues tested. In adult tissues, the expression levels of Pum1 and Pum2 were expressed at higher levels in the testis and muscle than in any other tissue. The characteristics of the tissue-specific expression of Pum genes suggest that Pum1 and Pum2 have effects crucially in particular stage during development of chicken gonads depending on sexual maturation.     

A comparison of BRCT domains involved in nonhomologous end-joining: introducing the solution structure of the BRCT domain of polymerase lambda

Three of the four family X polymerases, DNA polymerase lambda, DNA polymerase mu, and TdT, have been associated with repair of double-strand DNA breaks by nonhomologous end-joining. Their involvement in this DNA repair process requires an N-terminal BRCT domain that mediates interaction with other protein factors required for recognition and binding of broken DNA ends. Here we present the NMR solution structure of the BRCT domain of DNA polymerase lambda, completing the structural portrait for this family of enzymes. Analysis of the overall fold of the polymerase lambda BRCT domain reveals structural similarity to the BRCT domains of polymerase mu and TdT, yet highlights some key sequence and structural differences that may account for important differences in the biological activities of these enzymes and their roles in nonhomologous end-joining. Mutagenesis studies indicate that the conserved Arg57 residue of Pol lambda plays a more critical role for binding to the XRCC4-Ligase IV complex than its structural homolog in Pol mu, Arg43. In contrast, the hydrophobic Leu60 residue of Pol lambda contributes less significantly to binding than the structurally homologous Phe46 residue of Pol mu. A third leucine residue involved in the binding and activity of Pol mu, is nonconservatively replaced by a glutamine in Pol lambda (Gln64) and, based on binding and activity data, is apparently unimportant for Pol lambda interactions with the NHEJ complex. In conclusion, both the structure of the Pol lambda BRCT domain and its mode of interaction with the other components of the NHEJ complex significantly differ from the two previously studied homologs, Pol mu and TdT.     

Mitochondrial ATP synthase is a target for TNBS-induced protein carbonylation in XS-106 dendritic cells

ROS are produced in dendritic cells (DCs) during antigen presentation in contact hypersensitivity (CHS). As a result, ROS cause a number of nonenzymatic protein modifications, including carbonylation, which is the most widely used marker of oxidative stress. 2,4,6-Trinitrobenzene sulfonic acid (TNBS) is a well-characterized contact allergen that results in the formation of ROS. However, proteins that are carbonylated in DCs in response to TNBS have not been identified. To study ROS-dependent protein carbonylation in response to TNBS, we used the well-established mouse DC line, XS-106. We focused on the effects of TNBS on oxidation by examining selected oxidative markers. We identified TNBS-induced ROS and myeloperoxidase (MPO) proteins and demonstrated that the increase in ROS resulted in IL-12 production. The increase in oxidation was further confirmed by an oxidation-dependent increase in protein modifications, such as carbonylation. In fact, TNBS strongly induced carbonylation of mitochondrial adenosine triphosphate (ATP) synthase in XS-106 DCs, as determined by MALDI-TOF analysis and 2-D Western blotting. ROS production and protein carbonylation were confirmed in human monocyte-derived DCs (Mo-DCs). Furthermore, glutathione (GSH) decreased ROS and protein carbonylation in Mo-DCs. Carbonylation of ATP synthase in DCs may contribute to the pathophysiology of CHS.     

Transduced human PEP-1-heat shock protein 27 efficiently protects against brain ischemic insult

Reactive oxygen species contribute to the development of various human diseases. Ischemia is characterized by both significant oxidative stress and characteristic changes in the antioxidant defense mechanism. Heat shock protein 27 (HSP27) has a potent ability to increase cell survival in response to oxidative stress. In the present study, we have investigated the protective effects of PEP-1-HSP27 against cell death and ischemic insults. When PEP-1-HSP27 fusion protein was added to the culture medium of astrocyte and primary neuronal cells, it rapidly entered the cells and protected them against cell death induced by oxidative stress. Immunohistochemical analysis revealed that, when PEP-1-HSP27 fusion protein was intraperitoneally injected into gerbils, it prevented neuronal cell death in the CA1 region of the hippocampus in response to transient forebrain ischemia. Our results demonstrate that transduced PEP-1-HSP27 protects against cell death in vitro and in vivo, and suggest that transduction of PEP-1-HSP27 fusion protein provides a potential strategy for therapeutic delivery in various human diseases in which reactive oxygen species are implicated, including stroke.     

High-resolution human papillomavirus genotyping by MALDI-TOF mass spectrometry

We describe a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS)-based assay for human papillomavirus (HPV) genotyping--the restriction fragment mass polymorphism (RFMP) assay, which is based on mass measurement of genotype-specific oligonucleotide fragments generated by TypeIIS restriction endonuclease cleavage after recognition sites have been introduced by PCR amplification. The use of a TypeIIS restriction enzyme makes the RFMP assay independent of sequence and applicable to a wide variety of HPV genotypes, because these enzymes have cleavage sites at a fixed distance from their recognition sites. After PCR amplification, samples are subjected to restriction enzyme digestion with FokI and BtsCI and desalting using Oasis purification plates, followed by analysis by MALDI-TOF MS. Overall, the protocol is simple, takes approximately 4-4.5 h and can accurately detect and identify at least 74 different HPV genotypes.     

Somatic embryogenesis and plant regeneration in elite genotypes of Picea koraiensis

Picea koraiensis, called Korean spruce, is an evergreen tree and found mostly in northeast Asia. In this study, plant regeneration via somatic embryogenesis from open-pollinated immature zygotic embryos of nine genotypes of elite trees was established. Immature zygotic embryos were cultured onto RJW medium modified from 505 medium with 21.48 μM NAA, 2.22 μM BA, and 2.32 μM KT. The average frequency for all nine genotypes was 74.2%. Embryogenic calluses of the nine genotypes of elite trees were subcultured on RJW basal medium containing 8.06 μM NAA, 1.11 μM BA, and 1.16 μM kinetin. The calluses of three lines, 3^#, 9^#, and 2^#, were actively proliferated but others were not. Somatic embryogenesis was induced from the embryogenic callus in genotypes of 3^#, 9^#, and 2^# on RJW medium with ABA and 60 g l⁻¹ sucrose. Cotyledonary somatic embryos were subjected to a drying process. The drying of embryos by uncapping the culture bottle for 5 days on a clean bench resulted in a high frequency of germination of somatic embryos (87% in RJW medium). However, plantlet conversion from germinated embryos was greatly reduced and the optimal medium for plant conversion was 1/2 WPM or 1/2 BMI medium. In conclusion, we have, for the first time, established a plant regeneration system via somatic embryogenesis in the Korean spruce, which can be applied for rapid micropropagation of elite trees.