Degradation of diazinon by 2,4-dihydroxy-7-methoxy-2h-1, 4-benzoxazin-3(4h)-one in maize

A cyclic hydroxamate, 2,4-dihydroxy-7-methoxy-2H- 1,4-benzoxazin-3(4H)-one (DIMBOA), was isolated and identified from shoots of 6-day-old corn seedlings grown in the dark. From 100 g of plant tissue 100 mg of DIMBOA were isolated. This hydroxamate was very effective in catalysing the hydrolysis of the pyrimidinyl organophosphate insecticide, diazinon (O, O-diethyl- O-[6- methyl-2-(1-methylethyl)-4-pyrimidinyl] phosphorothioate) to 6- methyl-2-(1-methylethyl)-4-hydroxypyrimidine and diethyl phosphorothioic acid. The optimum pH for hydrolytic activity was 5 and at pH values equal to or higher than the pK_a of the hydroxamic group (6.95) most of the activity was lost.     

Biological Activity of Nerve Growth Factor Precursor Is Dependent upon Relative Levels of Its Receptors

Nerve growth factor (NGF) is produced as a precursor called pro-nerve growth factor (proNGF), which is secreted by many tissues and is the predominant form of NGF in the central nervous system. In Alzheimer disease brain, cholinergic neurons degenerate and can no longer transport NGF as efficiently, leading to an increase in untransported NGF in the target tissue. The protein that accumulates in the target tissue is proNGF, not the mature form. The role of this precursor is controversial, and both neurotrophic and apoptotic activities have been reported for recombinant proNGFs. Differences in the protein structures, protein expression systems, methods used for protein purification, and methods used for bioassay may affect the activity of these proteins. Here, we show that proNGF is neurotrophic regardless of mutations or tags, and no matter how it is purified or in which system it is expressed. However, although proNGF is neurotrophic under our assay conditions for primary sympathetic neurons and for pheochromocytoma (PC12) cells, it is apoptotic for unprimed PC12 cells when they are deprived of serum. The ratio of tropomyosin-related kinase A to p75 neurotrophin receptor is low in unprimed PC12 cells compared with primed PC12 cells and sympathetic neurons, altering the balance of proNGF-induced signaling to favor apoptosis. We conclude that the relative level of proNGF receptors determines whether this precursor exhibits neurotrophic or apoptotic activity.Nerve growth factor (NGF)³ regulates neuronal survival, neurite outgrowth, and differentiation in the peripheral and central nervous systems (1). The mature form of NGF forms a non-covalent homodimer and binds with high affinity (k_d ≈ 10⁻¹¹ m) to tropomyosin-related kinase A (TrkA) and with low affinity (k_d ≈ 10⁻⁹ m) to the common neurotrophin receptor p75^NTR (p75 neurotrophin receptor) (2). NGF promotes cell survival and growth in cells expressing TrkA through activation of the phosphatidylinositol 3-kinase/AKT pathway and the Ras/mitogen-activated protein kinase (MAPK) pathway (3, 4). p75^NTR plays diverse roles, ranging from cell survival to cell death depending on the cellular context in which it is expressed. Through activation of the NF-κB pathway, p75^NTR can contribute to cell survival in sensory neurons (5), it is involved in axonal growth via regulation of Rho activity (6), and it can interact with Trks to enhance neurotrophin affinity (at low concentration of ligand) and specificity of binding to Trks (7–9). High levels of p75^NTR expression can induce apoptosis when there are low levels of Trk or when Trk is absent (10, 11). Apoptosis occurs through increased ceramide production (12), activation of c-Jun N-terminal kinase (JNK1), and p53 (10, 13). p75^NTR requires a co-receptor called sortilin to induce cell death (14).NGF is produced as a precursor called pro-nerve growth factor (proNGF) (15). ProNGF is secreted by many tissues such as prostate cells, spermatids, hair follicles, oral mucosal keratinocytes, sympathetic neurons, cortical astrocytes, heart, and spleen (16–20). ProNGF is the predominant form of NGF in the central and peripheral nervous systems, whereas little or no mature NGF can be detected (21–24). In Alzheimer disease brain, retrograde transport from the cortex and hippocampus to basal forebrain cholinergic neurons is reduced as these neurons degenerate, with concomitant proNGF accumulation in the cortex and hippocampus (21, 23). This suggested that proNGF mediates biological activity besides its prodomain function of promoting protein folding and regulation of neurotrophin secretion (25–28). To study the role of proNGF protein in vitro, point mutations were inserted at the cleavage site used by furin, a proprotein convertase known to cleave proNGF (29), to minimize the conversion of proNGF to mature NGF. The resulting recombinant, cleavage-resistant proNGFs reportedly exhibit either apoptotic activity (30, 31) or neurotrophic activity (32, 33). These recombinant proteins differ in several ways (

Helicase binding to DnaI exposes a cryptic DNA-binding site during helicase loading in Bacillus subtilis

The Bacillus subtilis DnaI, DnaB and DnaD proteins load the replicative ring helicase DnaC onto DNA during priming of DNA replication. Here we show that DnaI consists of a C-terminal domain (Cd) with ATPase and DNA-binding activities and an N-terminal domain (Nd) that interacts with the replicative ring helicase. A Zn²⁺-binding module mediates the interaction with the helicase and C67, C70 and H84 are involved in the coordination of the Zn²⁺. DnaI binds ATP and exhibits ATPase activity that is not stimulated by ssDNA, because the DNA-binding site on Cd is masked by Nd. The ATPase activity resides on the Cd domain and when detached from the Nd domain, it becomes sensitive to stimulation by ssDNA because its cryptic DNA-binding site is exposed. Therefore, Nd acts as a molecular ‘switch’ regulating access to the ssDNA binding site on Cd, in response to binding of the helicase. DnaI is sufficient to load the replicative helicase from a complex with six DnaI molecules, so there is no requirement for a dual helicase loader system.     

The social context of cannibalism in migratory bands of the Mormon cricket

Cannibalism has been shown to be important to the collective motion of mass migratory bands of insects, such as locusts and Mormon crickets. These mobile groups consist of millions of individuals and are highly destructive to vegetation. Individuals move in response to attacks from approaching conspecifics and bite those ahead, resulting in further movement and encounters with others. Despite the importance of cannibalism, the way in which individuals make attack decisions and how the social context affects these cannibalistic interactions is unknown. This can be understood by examining the decisions made by individuals in response to others. We performed a field investigation which shows that adult Mormon crickets were more likely to approach and attack a stationary cricket that was side-on to the flow than either head- or abdomen-on, suggesting that individuals could reduce their risk of an attack by aligning with neighbours. We found strong social effects on cannibalistic behaviour: encounters lasted longer, were more likely to result in an attack, and attacks were more likely to be successful if other individuals were present around a stationary individual. This local aggregation appears to be driven by positive feedback whereby the presence of individuals attracts others, which can lead to further crowding. This work improves our understanding of the local social dynamics driving migratory band formation, maintenance and movement at the population level.     

A high yield procedure for the preparation of arsonolipids (2,3-diacyloxypropylarsonic acids)

The crucial step in the preparation of the title arsonolipids starting from the dichloromethane-soluble dithioarsonite CH₂(OH)CH(OH)CH₂–As(SPh)₂ is to avoid an internal cyclization during the acylation which protects the primary –OH group from being acylated. This was to a large extent accomplished by using fatty acyl chloride in the presence of the weak base pyridine and controlling the temperature and rate of the acyl chloride addition, giving ～70% yields of arsonolipids. The presence of catalytic amounts of 4-dimethylaminopyridine boosted the yields to 82–85%. This yield is a great improvement over the yields (20–55%) previously achieved. The acylating systems (RCO)₂O or RCOCl and BF₃·Et₂O gave only moderate yields (25–60%) of arsonolipids.     

Pharmacological Screening Using an FXN-EGFP Cellular Genomic Reporter Assay for the Therapy of Friedreich Ataxia

Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol.     

Dietary cholesterol exacerbates hepatic steatosis and inflammation in obese LDL receptor-deficient mice

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, can progress to steatohepatitis (NASH) and advanced liver disease. Mechanisms that underlie this progression remain poorly understood, partly due to lack of good animal models that resemble human NASH. We previously showed that several metabolic syndrome features that develop in LDL receptor-deficient (LDLR-/-) mice fed a diabetogenic diet are worsened by dietary cholesterol. To test whether dietary cholesterol can alter the hepatic phenotype in the metabolic syndrome, we fed LDLR-/- mice a high-fat, high-carbohydrate diabetogenic diet (DD) without or with added cholesterol (DDC). Both groups of mice developed obesity and insulin resistance. Hyperinsulinemia, dyslipidemia, hepatic triglyceride, and alanine aminotransferase (ALT) elevations were greater with DDC. Livers of DD-fed mice showed histological changes resembling NAFLD, including steatosis and modest fibrotic changes; however, DDC-fed animals developed micro- and macrovesicular steatosis, inflammatory cell foci, and fibrosis resembling human NASH. Dietary cholesterol also exacerbated hepatic macrophage infiltration, apoptosis, and oxidative stress. Thus, LDLR-/- mice fed diabetogenic diets may be useful models for studying human NASH. Dietary cholesterol appears to confer a second "hit" that results in a distinct hepatic phenotype characterized by increased inflammation and oxidative stress.