Coordination chemistry of co(II)-bleomycin: its investigation through NMR and molecular dynamics

Previous studies on the coordination chemistry of Co-bleomycin have suggested the secondary amine in beta-aminoalanine, the N5 and N1 nitrogens in the pyrimidine and imidazole rings, respectively, and the amide nitrogen in beta-hydroxyhistidine as equatorial ligands to the cobalt ion. The primary amine in beta-aminoalanine and the carbamoyl group of the mannose have been proposed alternatively as possible axial ligands. The first coordination sphere of Co(II) in Co(II)BLM has been investigated in the present study through the use of NMR and molecular dynamics calculations. The data collected from the NMR experiments are in agreement with the equatorial ligands previously proposed, and also support the participation of the primary amine as an axial ligand. The paramagnetic shifts of the gulose and mannose protons could suggest the latter as a second axial ligand. This possibility was investigated by way of molecular dynamics, with distance restraints derived from the relaxation times measured through NMR. The molecular dynamics results indicate that the most favorable structure is six-coordinate, with the primary amine and either the carbamoyl oxygen or a solvent molecule occupying the axial sites. The analysis of the structures previously derived for HOO-Co(III)-bleomycin and HOO-Co(III)-pepleomycin led us to propose the six-coordinate structure with only endogenous ligands, as the one held in solution by the Co(II) derivative of bleomycin.     

Do You Really Want to Know? Elementary Music Personnel and Potential in Utah

This is the second of two articles reporting the results of a study by the author regarding the status of elementary music education in the state of Utah. This article focuses on the qualifications of Utah's elementary music teachers (music certified, elementary classroom certified, artists-in-residence, volunteers, and paraprofessionals) and the conditions under which they teach. Interactions among teacher qualifications and teaching status are explored. Paraprofessionals play a significant role in Utah's elementary music programs. While over 90 percent of elementary schools in the United States provide regular music instruction taught by certified music specialists, less than 10 percent of Utah's elementary population receives such instruction. Nearly half of the elementary students in the state receive no designated music instruction beyond that provided by their regular elementary classroom teacher. The influence of school funding, No Child Left Behind and other accountability measures, high-stakes testing, urban/rural populations, and leadership are highlighted. Policy considerations are discussed.     

Free fatty acid-induced hepatic insulin resistance: a potential role for protein kinase C-delta

The mechanisms of the impairment in hepatic glucose metabolism induced by free fatty acids (FFAs) and the importance of FFA oxidation in these mechanisms remain unclear. FFA-induced peripheral insulin resistance has been linked to membrane translocation of novel protein kinase C (PKC) isoforms, but the role of PKC in hepatic insulin resistance has not been assessed. To investigate the biochemical pathways that are induced by FFA in the liver and their relation to glucose metabolism in vivo, we determined endogenous glucose production (EGP), the hepatic content of citrate (product of acetyl-CoA derived from FFA oxidation and oxaloacetate), and hepatic PKC isoform translocation after 2 and 7 h Intralipid + heparin (IH) or SAL in rats. Experiments were performed in the basal state and during hyperinsulinemic clamps (insulin infusion rate, 5 mU. kg(-1). min(-1)). IH increased EGP in the basal state (P < 0.001) and during hyperinsulinemia (P < 0.001) at 2 and 7 h. Also, 7-h infusion of IH induced resistance to the suppressive effect of insulin on EGP (P < 0.05). Glycerol infusion (resulting in plasma glycerol levels similar to IH infusion) did not have any effect on EGP. IH increased hepatic citrate content by twofold, independent of the insulin levels and the duration of IH infusion. IH induced hepatic PKC-delta translocation from the cytosolic to membrane fraction in all groups. PKC-delta translocation was greater at 7 compared with 2 h (P < 0.05). In conclusion, 1) increased FFA oxidation may contribute to the FFA-induced increase in EGP in the basal state and during hyperinsulinemia but is not associated with FFA-induced hepatic insulin resistance, and 2) the progressive insulin resistance induced by FFA in the liver is associated with a progressive increase in hepatic PKC-delta translocation.     

Electron transfer associated with oxygen activation in the B2 protein of ribonucleotide reductase from Escherichia coli

Each of the two beta peptides which comprise the B2 protein of Escherichia coli ribonucleotide reductase (RRB2) possesses a nonheme dinuclear iron cluster and a tyrosine residue at position 122. The oxidized form of the protein contains all high spin ferric iron and 1.0-1.4 tyrosyl radicals per RRB2 protein. In order to define the stoichiometry of in vitro dioxygen reduction catalyzed by fully reduced RRB2 we have quantified the reactants and products in the aerobic addition of Fe(II) to metal-free RRB2apo utilizing an oxygraph to quantify oxygen consumption, electron paramagnetic resonance to measure tyrosine radical generation, and M?ssbauer spectroscopy to determine the extent of iron oxidation. Our data indicate that 3.1 Fe(II) and 0.8 Tyr122 are oxidized per mol of O2 reduced. M?ssbauer experiments indicate that less than 8% of the iron is bound as mononuclear high spin Fe(III). Further, the aerobic addition of substoichiometric amounts of 57Fe to RRB2apo consistently produces dinuclear clusters, rather than mononuclear Fe(III) species, providing the first direct spectroscopic evidence for the preferential formation of the dinuclear units at the active site. These stoichiometry studies were extended to include the phenylalanine mutant protein (Y122F)RRB2 and show that 3.9 mol-equivalents of Fe(II) are oxidized per mol of O2 consumed. Our stoichiometry data has led us to propose a model for dioxygen activation catalyzed by RRB2 which invokes electron transfer between iron clusters.     

Activated Notch Causes Deafness by Promoting a Supporting Cell Phenotype in Developing Auditory Hair Cells

Purpose

To determine whether activated Notch can promote a supporting cell fate during sensory cell differentiation in the inner ear.

Methods

An activated form of the Notch1 receptor (NICD) was expressed in early differentiating hair cells using a Gfi1-Cre mouse allele. To determine the effects of activated Notch on developing hair cells, Gfi1-NICD animals and their littermate controls were assessed at 5 weeks for hearing by measuring auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). The differentiation of NICD-expressing hair cells was assessed at postnatal day (P) 6, 11 and 20, using histological and molecular markers for hair cells, as well as supporting cells/progenitor cells. We also examined whether the effects of Notch were mediated by SOX2, a gene expressed in supporting cells and a likely downstream target of Notch, by crossing an inducible form of SOX2 to the Gfi1-Cre.

Results

Activation of Notch1 in developing auditory hair cells causes profound deafness. The NICD-expressing hair cells switch off a number of hair cell markers and lose their characteristic morphology. Instead, NICD-expressing hair cells adopt a morphology resembling supporting cells and upregulate a number of supporting cell markers. These effects do not appear to be mediated by SOX2, because although expression of SOX2 caused some hearing impairment, the SOX2-expressing hair cells did not downregulate hair cell markers nor exhibit a supporting cell-like phenotype.

Conclusions

Our data show that Notch signaling inhibits hair cell differentiation and promotes a supporting cell-like phenotype, and that these effects are unlikely to be mediated by SOX2.     

Linking responses of native and invasive plants to hurricane disturbances: implications for coastal plant community structure

Hurricane disturbances produce significant changes in forest microclimates, creating opportunities for seedling regeneration of native and invasive plant species alike. However, there is limited information on how changes in microclimates and pre-existing forest conditions affect native and invasive plants responses to hurricane disturbances. In this manipulative study, we examined the responses of three common shrub/small stature tree species, two of which are native to the coastal region of the southeastern USA (Baccharis halimifolia and Morella cerifera) and one that is invasive (Triadica sebifera), to two key components of hurricane disturbance (canopy damage and saline storm surge). In a greenhouse, we grew seedlings of these species under a range of shade levels that mimicked pre-and post-hurricane canopy conditions for wet pine forest and mixed hardwood forest, two forest communities common in coastal areas of the southeastern USA. Seedlings were subjected to saline storm surges equivalent to full strength sea water for 3 days. Seedling responses (mortality and growth) to the treatments were monitored for 16 months. All species benefitted from higher canopy openness. Storm surge effects were short-lived and seedlings readily recovered under high light conditions. The storm surge had stronger negative effects on survival and growth of all species when coupled with high shade, suggesting storm surge has greater negative impacts on seedlings where hurricane winds cause minimal or no canopy damage. The invasive T. sebifera was by far more shade tolerant than the natives. Survival of T. sebifera seedlings under highly shaded conditions may provide it a competitive edge over native species during community reassembly following tropical storms. Differential responses of native and invasive species to hurricane disturbances will have profound consequences on community structure across coastal forest stands, and may be regulated by legacies of prior disturbances, community structure, extent of canopy damage, and species’ tolerance to specific microclimates.

     

转基因大豆非同源对照模板QC-PCR检测方法的建立

建立了转基因大豆CP4EPSPS基因的非同源对照模板QC-PCR检测方法。非同源竞争对照构建方法如下:利用CP4EPSPS基因特异引物,以大肠杆菌基因组DNA为异源模板,在低严谨度PCR扩增,回收适宜DNA片段并克隆到pMD-8载体上。该片段与CP4EPSPS基因相应序列除两端引物序列完全相同外,其他部分没有同源性。