Solvent cage effects: the influence of radical mass and volume on the recombination dynamics of radical cage pairs generated by photolysis of [CpCH2CH2N(CH3)C(O)(CH2)nCH3Mo(CO)3]2 (n = 3, 8, 13, 18) (Cp = eta 5-C5H4) complexes

The photochemistry of the [(CpR)Mo(CO)(3)](2) molecules, where CpR = eta(5)-C(5)H(4)(CH(2))(2)C(O)NCH(3)(CH(2))(n)CH(3) (n = 3, 8, 13, and 18), was examined using femtosecond pump-probe transient absorption spectroscopy. The goal of this study was to investigate the importance of radical size and mass on the dynamics and efficiency of geminate radical-radical recombination. The femtosecond results demonstrated the lack of any size/mass dependence of the recombination efficiency. This finding contrasts with results from a prior study that did find a size/mass dependence using a steady-state photochemical technique. To explain these conflicting results, it is proposed that the femtosecond pump-probe results are a measurement of the efficiency of primary geminate recombination whereas the steady-state method results are a measurement of the sum of primary and secondary geminate recombination efficiencies. The size/mass dependence is evident in the latter because secondary geminate recombination is a slower diffusive recombination process and therefore depends on the steric properties of the radicals. Although the existence of primary and secondary recombination channels is often taken for granted, experimental differentiation of primary and secondary caging has proven to be difficult because it is not possible for a single experimental technique to span the entire timescale for recombination of a radical cage pair and adequately resolve these recombination pathways.     

Isolation of phosphorylated polysaccharides from algae: the immunostimulatory principle of Chlorella pyrenoidosa

The major immunostimulatory principle in the hot aqueous extract of Chlorella pyrenoidosa has been isolated by a sequence of ethanol precipitation, precipitation with a cationic surfactant (CTAB), size exclusion chromatography, and anion exchange chromatography. A series of phosphorylated polysaccharides were obtained having different molecular masses but with similar structures. The higher molecular mass fractions showed considerable activity in the stimulation of mouse peritoneal macrophages to synthesize nitric oxide. The structure of the major polysaccharide was established by sugar analysis, configurational analysis, and 1D and 2D NMR experiments at 500 and 800 MHz on the parent polysaccharide, the de-O-acetylated polysaccharide, and on the components obtained after hydrolysis of the phosphate diesters. It had a β-d-Galp-(1→3)-β-d-Galp-(1→3)-backbone with half of the Galp units substituted at O-6 by terminal β-d-Glcp units. The remaining Galp units were substituted on O-6 by about equal amounts of α-d-Manp-1-phosphate and 3-O-Me-α-Manp-1-phosphate diesters. The substituents were not located in a regularly alternating fashion on the backbone. The O-acetyl groups were largely located on O-2 and O-4 of Galp and 35% of the Galp residues were O-acetylated. This is the second observation of a phosphorylated polysaccharide in an alga and the first where it is present to a significant extent.     

Neuronal Basis of Innate Olfactory Attraction to Ethanol in Drosophila

The decision to move towards a mating partner or a food source is essential for life. The mechanisms underlying these behaviors are not well understood. Here, we investigated the role of octopamine – the invertebrate analogue of noradrenaline – in innate olfactory attraction to ethanol. We confirmed that preference is caused via an olfactory stimulus by dissecting the function of the olfactory co-receptor Orco (formally known as OR83b). Orco function is not required for ethanol recognition per se, however it plays a role in context dependent recognition of ethanol. Odor-evoked ethanol preference requires the function of Tbh (Tyramine β hydroxalyse), the rate-limiting enzyme of octopamine synthesis. In addition, neuronal activity in a subset of octopaminergic neurons is necessary for olfactory ethanol preference. Notably, a specific neuronal activation pattern of tyraminergic/octopaminergic neurons elicit preference and is therefore sufficient to induce preference. In contrast, dopamine dependent increase in locomotor activity is not sufficient for olfactory ethanol preference. Consistent with the role of noradrenaline in mammalian drug induced rewards, we provide evidence that in adult Drosophila the octopaminergic neurotransmitter functions as a reinforcer and that the molecular dissection of the innate attraction to ethanol uncovers the basic properties of a response selection system.     

Oxidative stress mediates neuronal DNA damage and apoptosis in response to cytosine arabinoside

Cytosine arabinoside (AraC) is a nucleoside analog that produces significant neurotoxicity in cancer patients. The mechanism by which AraC causes neuronal death is a matter of some debate because the conventional understanding of AraC toxicity requires incorporation into newly synthesized DNA. Here we demonstrate that AraC-induced apoptosis of cultured cerebral cortical neurons is mediated by oxidative stress. AraC-induced cell death was reduced by treatment with several different free-radical scavengers (N-acetyl-L-cysteine, dipyridamole, uric acid, and vitamin E) and was increased following depletion of cellular glutathione stores. AraC induced the formation of reactive oxygen species in neurons as measured by an increase in the fluorescence of the dye 5-(6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate. AraC produced DNA single-strand breaks as measured by single-cell gel electrophoresis and the level of DNA strand breakage was reduced by treatment with the free radical scavengers. These data support a model in which AraC induces neuronal apoptosis by provoking the generation of reactive oxygen species, causing oxidative DNA damage and initiating the p53-dependent apoptotic program. These observations suggest the use of antioxidant therapies to reduce neurotoxicity in AraC chemotherapeutic regimens.     

A cluster of Ankyrin and Ankyrin-TPR repeat genes is associated with panicle branching diversity in rice

The number of grains per panicle is an important yield-related trait in cereals which depends in part on panicle branching complexity. One component of this complexity is the number of secondary branches per panicle. Previously, a GWAS site associated with secondary branch and spikelet numbers per panicle in rice was identified. Here we combined gene capture, bi-parental genetic population analysis, expression profiling and transgenic approaches in order to investigate the functional significance of a cluster of 6 ANK and ANK-TPR genes within the QTL. Four of the ANK and ANK-TPR genes present a differential expression associated with panicle secondary branch number in contrasted accessions. These differential expression patterns correlate in the different alleles of these genes with specific deletions of potential cis-regulatory sequences in their promoters. Two of these genes were confirmed through functional analysis as playing a role in the control of panicle architecture. Our findings indicate that secondary branching diversity in the rice panicle is governed in part by differentially expressed genes within this cluster encoding ANK and ANK-TPR domain proteins that may act as positive or negative regulators of panicle meristem’s identity transition from indeterminate to determinate state.     

Ectoine accumulation in Brevibacterium epidermis

As a halotolerant bacterial species, Brevibacterium epidermis DSM 20659 can grow at relatively high salinity, tolerating up to 2 m NaCl. It synthesizes ectoine and the intracellular content increases with the medium salinity, with a maximum of 0.14 g ectoine/g CDW at 1 m NaCl. Sugar-stressed cells do not synthesize ectoine. Ectoine synthesis is also affected by the presence of external osmolytes. Added betaine is taken up and completely replaced ectoine, while l-proline is only temporarily accumulated after which ectoine is synthesized. The strain can metabolize ectoine; l-glutamate is a better carbon source for ectoine synthesis than l-aspartate.     

Revisited and large-scale synthesis and purification of the mutated and wild type neu/erbB-2 membrane-spanning segment.

Solid-phase syntheses of the hydrophobic peptides Neu(TM35) ((1)EQRASPVTFIIATVVGVLLFLILVVVVGILIKRRR(35)) and Neu*(TM35) ((1)EQRASPVTFIIATVEGVLLFLILVVVVGILIKRRR(35)), corresponding to the native and mutated (V15E) transmembrane domain of the neu/erbB-2 tyrosine kinase receptor, respectively, were accomplished using Fmoc chemistry. The use of a new resin and cleavage and purification conditions led to large increases in yields and peptide purity. Two (15)N-labelled versions of both wild type and mutated peptides were also synthesized. Approximately 20-40 mg of peptide was obtained using a small-scale synthesis, whereas ca 100 mg of pure peptide was collected on a medium scale. Peptide purity, as monitored by HPLC and mass spectrometry, ranged from 95 to 98% for the six peptides synthesized. Secondary structure as determined by UV circular dichroism (CD) in trifluoroethanol (TFE) showed ca 74% alpha-helical content for the native peptide and ca 63% for that bearing the mutation. Secondary structure of Neu(TM35) was retained in DMPC (dimyristoylphosphatidylcholine)/DCPC (dicaproylphosphatidylcholine) membrane bicelles, and evidences for dimers/oligomers in the lipid bilayer were found.