On the mechanisms of the reaction of dodecatungstophosphate with alkyl radicals in aqueous solutions

The reduced lacunary polyoxotungstate, [PW₁₁O₃₉]⁸⁻, reacts with the ^.CH₂CH(OH)CH₃ and ^.CH₂C(CH₃)₂OH radicals via a mechanism involving β-hydroxide elimination to yield propene and 2-methyl propene respectively, and [PW₁₁O₃₉]⁷⁻. [PW₁₁O₃₉]⁸⁻ is also oxidized by methyl radicals in a reaction which yields methane as the major product. It is proposed that the reactions proceed via the formation of short lived transients with W-C σ bonds.     

Proteins at the Polypeptide Tunnel Exit of the Yeast Mitochondrial Ribosome

Oxidative phosphorylation in mitochondria requires the synthesis of proteins encoded in the mitochondrial DNA. The mitochondrial translation machinery differs significantly from that of the bacterial ancestor of the organelle. This is especially evident from many mitochondria-specific ribosomal proteins. An important site of the ribosome is the polypeptide tunnel exit. Here, nascent chains are exposed to an aqueous environment for the first time. Many biogenesis factors interact with the tunnel exit of pro- and eukaryotic ribosomes to help the newly synthesized proteins to mature. To date, nothing is known about the organization of the tunnel exit of mitochondrial ribosomes. We therefore undertook a comprehensive approach to determine the composition of the yeast mitochondrial ribosomal tunnel exit. Mitochondria contain homologues of the ribosomal proteins located at this site in bacterial ribosomes. Here, we identified proteins located in their proximity by chemical cross-linking and mass spectrometry. Our analysis revealed a complex network of interacting proteins including proteins and protein domains specific to mitochondrial ribosomes. This network includes Mba1, the membrane-bound ribosome receptor of the inner membrane, as well as Mrpl3, Mrpl13, and Mrpl27, which constitute ribosomal proteins exclusively found in mitochondria. This unique architecture of the tunnel exit is presumably an adaptation of the translation system to the specific requirements of the organelle.     

In ovo exposure to monochromatic green light promotes skeletal muscle cell proliferation and affects myofiber growth in posthatch chicks

Our previous studies demonstrated that illumination of chicken embryos with monochromatic green light results in enhanced body and muscle weight at later posthatch stages. In the present study, we investigated the cellular and molecular basis of this phenomenon. First, we showed that on day 6 posthatch, myofibers were more uniform in the in ovo illuminated group than in the control group incubated in the dark, with respect to the number of myofibers displaying diameter values within the range of the mean value. Second, we tested the hypothesis that in ovo illumination causes an increase in the number of myoblasts; this in turn can promote posthatch muscle growth. Indeed, a significant increase in the number of skeletal muscle cells isolated from pectoralis muscle was observed in the in ovo illuminated group on days 1 and 3 posthatch relative to the control group. This increased cell number was accompanied by higher expression levels of Pax7 and myogenin proteins on posthatch days 1 and 3, respectively. A parallel analysis of proliferating cells in the intact muscle further demonstrated a significant increase in the number of cells positive for proliferating cell nuclear antigen in muscle from the in ovo illuminated group. Third, we demonstrated that the transition from fetal- to adult-type myoblasts, normally occurring in late stages of chicken embryogenesis, is initiated earlier in embryos subjected to in ovo green-light illumination. We suggest that the stimulatory effect of in ovo illumination on posthatch muscle growth is the result of enhanced proliferation and differentiation of adult myoblasts and myofiber synchronization.     

The Drosophila caspase Ice is important for many apoptotic cell deaths and for spermatid individualization, a nonapoptotic process

Caspase family proteases play important roles in the regulation of apoptotic cell death. Initiator caspases are activated in response to death stimuli, and they transduce and amplify these signals by cleaving and thereby activating effector caspases. In Drosophila, the initiator caspase Nc (previously Dronc) cleaves and activates two short-prodomain caspases, Dcp-1 and Ice (previously Drice), suggesting these as candidate effectors of Nc killing activity. dcp-1-null mutants are healthy and possess few defects in normally occurring cell death. To explore roles for Ice in cell death, we generated and characterized an Ice null mutant. Animals lacking Ice show a number of defects in cell death, including those that occur during embryonic development, as well as during formation of adult eyes, arista and wings. Ice mutants exhibit subtle defects in the destruction of larval tissues, and do not prevent destruction of salivary glands during metamorphosis. Cells from Ice animals are also markedly resistant to several stresses, including X-irradiation and inhibition of protein synthesis. Mutations in Ice also suppress cell death that is induced by expression of Rpr, Wrinkled (previously Hid) and Grim. These observations demonstrate that Ice plays an important non-redundant role as a cell death effector. Finally, we demonstrate that Ice participates in, but is not absolutely required for, the non-apoptotic process of spermatid differentiation.     

Endothelin Signaling Pathways in Rat Adrenal Medulla

1. We further characterized the effect of endothelins (ETs) on receptor-mediated phosphoinositide (PI) turnover, nitric oxide synthase (NOS) activation, and cGMP formation in whole rat adrenal medulla.2. The PI hydrolysis was assessed as accumulation of inositol monophosphates (InsP₁) in the presence of 10 mM LiCl in whole tissue and the analysis of inositol-1-phosphate by Dowex anion exchange chromatography. NOS activity was assayed by monitoring the conversion of radiolabeled L-arginine to L-citrulline. Cyclic GMP formation was assessed as accumulation of cGMP in whole tissue in the presence of phosphodiesterase inhibition, and the amount of cGMP formed was determined by radioimmuno-antibody procedure.3. ET-1 and ET-3 increased PI turnover by 30% in whole adrenal medulla prelabeled with [³H] myoinositol. Both ETs isoforms, at equimolar doses, increased NOS activity and cGMP levels in similar degree. The selective ET_B receptor agonist, IRL-1620, also increased cGMP formation, mimicking the effects of ETs, while IRL-1620 did not alter the PI metabolism. ETs-induced InsP₁ accumulation and cGMP was dependent on extracellular calcium. The effect of ETs on PI turnover was inhibited by neomycin. The L-arginine analogue, N-nitro-L-arginine (L-NAME), and two inhibitors of soluble guanylyl cyclase, methylene blue and ODQ, significantly inhibited the increase in cGMP production induced by ETs or IRL-1620. The selective ET_A receptor antagonist, BQ 123, inhibited the ETs-induced increase in PI turnover, while the selective ET_B receptor antagonist, BQ 788, was ineffective. Likewise, BQ 788, significantly inhibited ET-1- or ET-3-induced NOS activation and cGMP generation but not ETs-induced InsP₁ accumulation.4. Our data indicate that stimulation of PI turnover and NO-induced cGMP generation constitutes ETs signaling pathways in rat adrenal medulla. The former action is mediated through activation of ET_A receptor, while the latter through the activation of ET_B receptor. These results support the role of endothelins in the regulation of adrenal medulla function.     

Nitrogen Transfer Between Plants: A 15N Natural Abundance Study with Crop and Weed Species

An increasing amount of evidence indicates that N can be transferred between plants. Nonetheless, a number of fundamental questions remain. A series of experiments was initiated in the field to examine N transfer between N₂-fixing soybean (Glycine max [L.] Merr.) varieties and a non-nodulating soybean, and between N₂-fixing peanut (Arachis hypogaea L.) or soybean and neighboring weed species. The experiments were conducted in soils with low N fertilities and used differences in N accumulation and/or ¹⁵N natural abundance to estimate N transfer. Mixtures of N₂-fixing and non-nod soybean indicated that substantial inter-plant N transfer occurred. Amounts were variable, ranging from negligible levels to 48% of the N found in the non-nod at maturity. Transfer did not appear to strongly penalize the N₂-fixing donor plants. But, in cases where high amounts of N were transferred, N content of donors was noticeably lowered. Differences were evident in the amount of N transferred from different N₂-fixing donor genotypes. Results of experiments with N₂-fixing crops and the weed species prickly sida (Sida spinosa L.) and sicklepod (Senna obtusifolia [L.] Irwin & Barneby) also indicated substantial N transfer occurred over a 60-day period, with amounts accounting for 30–80% of the N present in the weeds. Transfer of N, however, was generally very low in weed species that are known to be non-hosts for arbuscular mycorrhizae (yellow nutsedge, Cyperus esculentus L. and Palmer amaranth, Amaranthus palmeri [S.] Watson). The results are consistent with the view that N transfer occurs primarily through mycorrhizal hyphal networks, and they reveal that N transfer may be a contributing factor to weed problems in N₂-fixing crops in low N fertility conditions.     

Inhibitors can activate proteases to catalyze the synthesis and hydrolysis of peptides

Competitive inhibitors can activate proteases (papain, trypsin, and cathepsin S) to catalyze the synthesis of peptide bonds and accelerate the hydrolysis of poor substrates (from 1 to 99%). Reaction mixtures contained intermediate molecules that were formed by the coupling of the inhibitor with the poor substrate. This and other findings suggest the following chain of events. Part of the binding energy of formation of the enzyme-inhibitor complex was used to activate the inhibitor, i.e., to form acyl-enzyme species with a high-energy bond (e.g., a thioester bond in the case of papain) required for coupling the inhibitor with the substrate to form the intermediate molecule. The latter was subjected to successive reactions which led to a stepwise degradation of the substrate, as well as to the regeneration of the inhibitor. One mole of the inhibitor could catalyze rapid hydrolysis of at least 53 mol of substrate. The intermediate molecules were the species undergoing rapid hydrolysis. Therefore, 1 mol of inhibitor was involved in the synthesis of 53 mol of intermediate molecules; i.e., the inhibitor functioned as a cofactor that catalyzed the synthesis of peptides. Thus, the binding energy of formation of the enzyme-inhibitor complex can be utilized to catalyze the synthesis of peptide bonds in the absence of an exogenous energy source (e.g., ATP).     

Characterization of the insertase for β-barrel proteins of the outer mitochondrial membrane

The TOB–SAM complex is an essential component of the mitochondrial outer membrane that mediates the insertion of β-barrel precursor proteins into the membrane. We report here its isolation and determine its size, composition, and structural organization. The complex from Neurospora crassa was composed of Tob55–Sam50, Tob38–Sam35, and Tob37–Sam37 in a stoichiometry of 1:1:1 and had a molecular mass of 140 kD. A very minor fraction of the purified complex was associated with one Mdm10 protein. Using molecular homology modeling for Tob55 and cryoelectron microscopy reconstructions of the TOB complex, we present a model of the TOB–SAM complex that integrates biochemical and structural data. We discuss our results and the structural model in the context of a possible mechanism of the TOB insertase.     

Peripubertal vitamin D(3) deficiency delays puberty and disrupts the estrous cycle in adult female mice

The mechanism(s) by which vitamin D(3) regulates female reproduction is minimally understood. We tested the hypothesis that peripubertal vitamin D(3) deficiency disrupts hypothalamic-pituitary-ovarian physiology. To test this hypothesis, we used wild-type mice and Cyp27b1 (the rate-limiting enzyme in the synthesis of 1,25-dihydroxyvitamin D(3)) null mice to study the effect of vitamin D(3) deficiency on puberty and reproductive physiology. At the time of weaning, mice were randomized to a vitamin D(3)-replete or -deficient diet supplemented with calcium. We assessed the age of vaginal opening and first estrus (puberty markers), gonadotropin levels, ovarian histology, ovarian responsiveness to exogenous gonadotropins, and estrous cyclicity. Peripubertal vitamin D(3) deficiency significantly delayed vaginal opening without affecting the number of GnRH-immunopositive neurons or estradiol-negative feedback on gonadotropin levels during diestrus. Young adult females maintained on a vitamin D(3)-deficient diet after puberty had arrested follicular development and prolonged estrous cycles characterized by extended periods of diestrus. Ovaries of vitamin D(3)-deficient Cyp27b1 null mice responded to exogenous gonadotropins and deposited significantly more oocytes into the oviducts than mice maintained on a vitamin D(3)-replete diet. Estrous cycles were restored when vitamin D(3)-deficient Cyp27b1 null young adult females were transferred to a vitamin D(3)-replete diet. This study is the first to demonstrate that peripubertal vitamin D(3) sufficiency is important for an appropriately timed pubertal transition and maintenance of normal female reproductive physiology. These data suggest vitamin D(3) is a key regulator of neuroendocrine and ovarian physiology.