The Influences of A Nitrogen Atom Position in Dinucleoside 2-,3-,4-Pyridylphosphonates on Fragmentation Patterns in Electrospray Ionization Multistage Tandem Mass Spectra

2-,3-,4-Pyridylphosphonates and their phosphonothioate congeners were analyzed by electrospray ionization multistage tandem mass spectrometry (ESI-MSⁿ). It was found that the fragmentation pathways of these compounds were not influenced to any detectable extent by the stereochemistry at the phosphorus centers but were sensitive to the position of a nitrogen atom in the pyridine ring of these compounds. Possible mechanisms for fragmentations of the investigated compounds are discussed in detail.

     

Enhanced Phase II Detoxification Contributes to Beneficial Effects of Dietary Restriction as Revealed by Multi-platform Metabolomics Studies

Dietary restriction (DR) has many beneficial effects, but the detailed metabolic mechanism remains largely unresolved. As diet is essentially related to metabolism, we investigated the metabolite profiles of urines from control and DR animals using NMR and LC/MS metabolomic approaches. Multivariate analysis presented distinctive metabolic profiles and marker signals from glucuronide and glycine conjugation pathways in the DR group. Broad profiling of the urine phase II metabolites with neutral loss scanning showed that levels of glucuronide and glycine conjugation metabolites were generally higher in the DR group. The up-regulation of phase II detoxification in the DR group was confirmed by mRNA and protein expression levels of uridinediphospho-glucuronosyltransferase and glycine-N-acyltransferase in actual liver tissues. Histopathology and serum biochemistry showed that DR was correlated with the beneficial effects of low levels of serum alanine transaminase and glycogen granules in liver. In addition, the Nuclear factor (erythroid-derived 2)-like 2 signaling pathway was shown to be up-regulated, providing a mechanistic clue regarding the enhanced phase II detoxification in liver tissue. Taken together, our metabolomic and biochemical studies provide a possible metabolic perspective for understanding the complex mechanism underlying the beneficial effects of DR.It has been known for more than 70 years that dietary restriction (DR)¹ can extend the life span and delay the onset of age-related diseases, based on an early rodent study showing such effects (1). However, not until the 1980s was DR recognized as a good model for studying the mechanism of or inhibitory measures for aging (2). So far, extensive studies employing model organisms such as yeasts, nematodes, fruit flies, and rodents have shown that DR has beneficial effects in most of the species studied (for a review, see Ref. 3). Most notably, a recent 20-year-long study showed that monkeys, the species closest to humans, also benefit from DR similarly (4). Although there has not been (or could not have been) a systematic study on the effects of DR on the human life span, several longitudinal studies strongly suggest that changes in dietary intake can affect the life span and/or disease-associated marker values greatly (5–7).This inverse correlation between dietary intake and long-term health strongly indicates that DR''s effects should involve metabolism, and that DR elicits the reorganization of metabolic pathways. It also seems quite natural that something we eat should affect the body''s metabolism. Despite this seemingly straightforward relationship between diet and metabolism, the mechanisms underlying the beneficial effects of DR are anything but simple. Intensive efforts, spanning decades, to understand the mechanisms of DR have identified several genes that might mediate the effects of DR, such as mTOR, IGF-1, AMPK, and SIRT1 (for a review, see Ref. 8). Still, most of them are involved in early nutrient-sensing steps, and specific metabolic pathways, especially those at the final steps actually responsible for the effects of DR, are largely unknown.This might be at least partially due to the fact that previous studies have focused mostly on genomic or proteomic changes induced by DR, instead of looking at changes in metabolism or metabolites directly. Metabolomics, which has gained much interest in recent years (9–11), might be a good alternative for addressing the mechanistic uncertainty of DR''s effects, with the direct profiling of metabolic changes elicited by environmental factors. In contrast to genomics or proteomics, which often employ DNA or proteins extracted from particular tissues, metabolomics studies mostly employ body fluids (i.e. urine or blood), which can reflect the metabolic status of multiple organs, enabling investigations at a more systemic level. In particular, urine has been used extensively to study the mechanism of external stimuli (i.e. drugs or toxic insults) at most major target organs, such as the lung, kidney, liver, or heart (12–18). Still, metabolomics studies of DR effects have been very limited. A few previous ones reported the changes in phenomenological urine metabolic markers with DR, without identification and/or validation of specific metabolic pathways reflected at the actual tissue or enzyme level (19, 20). Therefore, those studies fell short of providing a mechanistic perspective on DR''s effects. In addition, they employed either NMR or LC/MS approaches without validation across the two analytical platforms.Among the metabolic pathways that can directly affect the integrity of multiple organs, and hence long-term health, are phase II detoxification pathways (21). Typically, lipophilic endo/xenobiotics are metabolized first by a phase I system, such as cytochrome P450, which modifies the compounds so that they have hydrophilic functional groups for increased solubility. In many cases, though, these modifications might increase the reactivity of the compounds, leading to cellular damage. The phase II detoxification systems involve conjugation reactions that attach charged hydrophilic molecular moieties to reactive metabolites, thus facilitating the elimination of the harmful metabolites from body, ultimately reducing their toxicity (22). These systems are thus especially important in protecting cellular macromolecules, such as DNA and proteins, from reactive electrophilic or nucleophilic metabolites. The enzymes involved in these processes include glutathione-S-transferase (GST), sulfotransferase, glycine-N-acyltransferase (GLYAT), and uridinediphospho-glucuronosyltransferase (UGT), with the last enzyme being the most prevalent (23). The beneficial effects of phase II reactions have been particularly studied in relation to the mechanism of healthy dietary ingredients. It is well believed that many such foods can prevent cancers (hence the term “chemoprevention”) by inducing phase II detoxification systems (24–26). Although DR also substantially reduces the incidence of cancers, the exact mechanism remains elusive.Here, we employed multi-platform metabolomics to obtain metabolic perspectives on the beneficial effects of DR on rats. Our results about urine metabolomics markers suggest that DR enhances the phase II detoxification pathway, which was confirmed by means of conjugation metabolite profiling and changes in mRNA/protein expression levels of phase II enzymes in actual liver tissues. A possible molecular mechanism was also addressed through the exploration of Nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) pathway activation upon DR. We believe the current study provides new metabolic insights into DR''s beneficial effects, as well as a workflow for studying DR''s effects from a metabolic perspective.     

Activated Cyclin-Dependent Kinase 5 Promotes Microglial Phagocytosis of Fibrillar β-Amyloid by Up-regulating Lipoprotein Lipase Expression

Amyloid plaques are crucial for the pathogenesis of Alzheimer disease (AD). Phagocytosis of fibrillar β-amyloid (Aβ) by activated microglia is essential for Aβ clearance in Alzheimer disease. However, the mechanism underlying Aβ clearance in the microglia remains unclear. In this study, we performed stable isotope labeling of amino acids in cultured cells for quantitative proteomics analysis to determine the changes in protein expression in BV2 microglia treated with or without Aβ. Among 2742 proteins identified, six were significantly up-regulated and seven were down-regulated by Aβ treatment. Bioinformatic analysis revealed strong over-representation of membrane proteins, including lipoprotein lipase (LPL), among proteins regulated by the Aβ stimulus. We verified that LPL expression increased at both mRNA and protein levels in response to Aβ treatment in BV2 microglia and primary microglial cells. Silencing of LPL reduced microglial phagocytosis of Aβ, but did not affect degradation of internalized Aβ. Importantly, we found that enhanced cyclin-dependent kinase 5 (CDK5) activity by increasing p35-to-p25 conversion contributed to LPL up-regulation and promoted Aβ phagocytosis in microglia, whereas inhibition of CDK5 reduced LPL expression and Aβ internalization. Furthermore, Aβ plaques was increased with reducing p25 and LPL level in APP/PS1 mouse brains, suggesting that CDK5/p25 signaling plays a crucial role in microglial phagocytosis of Aβ. In summary, our findings reveal a potential role of the CDK5/p25-LPL signaling pathway in Aβ phagocytosis by microglia and provide a new insight into the molecular pathogenesis of Alzheimer disease.Alzheimer disease (AD)¹ is one of the most common neurodegenerative disorders, which is characterized by pathological hallmarks such as neuronal and synaptic loss, neurofibrillary tangles (NFTs), and senile plaques. The intracellular NFTs are mainly composed of hyper-phosphorylated microtubule-associated protein tau, whereas toxic fibrillar β-amyloid (fAβ) as the main component of senile plaques is generated by sequential proteolytic cleavage of trans-membrane β-amyloid precursor protein (APP) by β- and γ-secretases. fAβ can induce oxidative stress-mediated neuronal cell death and cause cognitive impairment in mouse brains (1). Many reports suggest that fAβ induces dysregulation of two pivotal kinases CDK5 (2, 3) and GSK-3 (4), which are crucial regulators of hyperphosphorylated tau and increased production of Aβ from APP, and thereby triggers the cascade of signal transduction events underlying neuronal cell death in AD pathogenesis.As the resident immune cells in the brain, microglia can be activated in response to fAβ and often accumulate around the amyloid deposits in the brains of AD patients. Activated microglia trigger the production of inflammatory factors, reactive oxygen species, and chemokines, which may cause neuronal cell death (5). Furthermore, increasing evidence supports that activated microglia exert a vital beneficial role in the clearance of Aβ by phagocytosis. Many receptors, including scavenger receptor A (SR-A) (6), scavenger receptor class B type I (SR-BI) (7), lipopolysaccharide receptor (CD14) (8), CD33 (9), B-class scavenger receptor CD36 (10), CD47 (11), β1 integrin (12), toll-like receptor 2 (TLR2) (13), and toll-like receptor 4 (TLR4) (14), have been implicated in microglial phagocytosis of fAβ via direct or indirect binding to Aβ. Microglial phagocytosis of fAβ is also regulated by proinflammatory cytokines (15) and chemokine receptor CX3CR1 (16). Farfara et al. reported that the γ-secretase component presenilin, which is responsible for APP cleavage and Aβ production in neurons, is important for microglial fAβ clearance, indicating a dual role for presenilin in neuronal cell death and microglial phagocytosis (17). In addition, accumulating evidence suggests a critical role of lipids and lipoproteins in microglial fAβ phagocytosis and clearance. Lee et al. reported that apolipoprotein E (ApoE) enhances fAβ trafficking and degradation, indicating a role of cholesterol in fAβ degradation (18). After internalization, fAβ is degraded through the lysosome pathway (19, 20). However, the mechanism underlying microglial internalization of fAβ remains unclear.Stable isotope labeling of amino acids in cell culture (SILAC) is an accurate and reproducible mass spectrometry-based quantitative proteomics approach for examining changes in protein expression or post-translational modifications at a large scale (21, 22). Here, we used the SILAC quantitative proteomics strategy to investigate changes in the protein levels in BV2 microglia treated with fAβ. We found that 6 proteins were up-regulated and 7 were down-regulated significantly by Aβ treatment. Interestingly, bioinformatic analysis revealed that most of these up- or down-regulated proteins, including lipoprotein lipase (LPL), were mainly distributed in the cell membrane. We verified that LPL was up-regulated at both gene and protein levels in BV2 and primary microglia in response to fAβ, thereby indicating its role in the microglial phagocytosis of Aβ. Importantly, we further demonstrated that CDK5, which is a critical serine/threonine kinase in the pathogenesis of AD, regulated the expression of LPL and played a critical role in Aβ phagocytosis of microglia. Moreover, we found that increase in the p35-to-p25 conversion contributed to the enhanced CDK5 activity under Aβ stimulus and played a vital role in regulation of LPL expression and microglial Aβ phagocytosis. Our results suggest a role of the CDK5/p25-LPL signaling pathway in Aβ phagocytosis of microglia and provide valuable information to understand the molecular mechanism underlying microglial fAβ phagocytosis.     

Enhanced catalytic ability of Candida rugosa lipase immobilized on pore-enlarged hollow silica microspheres and cross-linked by modified dextran in both aqueous and non-aqueous phases

Candida rugosa lipase (CRL) is one of the most widely used lipases. To enhance the catalytic abilities of CRL in both aqueous and non-aqueous phases, hollow silica microspheres (HSMSs) with a pore size of 18.07 nm were used as an immobilization support, and aldehydecontaining dextrans were employed to further cross-link the adsorbed CRL. In the experimental ranges examined, the loading amount of lipase linearly increased to 171 ± 3.4 mg_protein/g_support with the CRL concentration and all the adsorption equilibriums were reached within 30 min. After simple cross-linking, the tolerance to pH 4.0 ～ 8.0 as well as the thermal stability of immobilized CRL at 40 ～ 80°C were both substantially increased, and 82 ± 2.1% activity remaining after the sixth reuse. The immobilized CRL was successfully applied to the resolution of racemic ibuprofen in non-aqueous phase. The initial reaction rate increased by 1.4- and 3.6-fold compared with the rates of adsorbed and native lipases, respectively. Furthermore, the R-ibuprofen was obtained at ee > 93%, and the enantiomeric ratio reached E > 140 at the conversion of 50 ± 1.5% within 48 h.     

Change of arthropod abundance in burned forests: Different patterns according to functional guilds

Forest fires are one of the most frequent and important causes of forest disturbances, the occurrence of which is globally increasing due to the effects of climate change. This study aimed to determine the impacts of fire and human activity on arthropod communities in affected forests. Twelve study sites in three burned areas were selected for this study. Intensities of disturbance in the study sites were characterized as follows: Disturbance Degree (DD) 0 (no fire), DD 1 (surface fire), DD 2 (crown fire), and DD 3 (crown fire followed by reforestation). Arthropods were collected using pitfall traps. Fourteen arthropod taxa (families, orders or classes), which are relatively homogeneous in their feeding habits and abundant, were analyzed. Depth of litter layer was selected as an environmental indicator for disturbance intensity, as it decreases linearly as the degree of disturbance increased. Changes of arthropod abundance in response to disturbance differed among functional guilds. As disturbance intensity increased, the abundance of detritivores decreased, but the abundance of herbivores increased. However, the abundance of predators varied between taxa. Formicidae and Araneae increased in disturbed sites, whereas Carabidae and Staphylinidae did not change. The abundance of Thysanura and Diptera was highly correlated with disturbance intensity, and may be suitable as a bioindicator for forest disturbance. Arthropod communities were more heterogeneous in forests of intermediate disturbance.     

Changes of butterfly communities after forest fire

Fires change the diversity and composition of insects in forest ecosystems. In the present study, we examined the change of butterfly communities after a fire including the increase of butterfly richness, grassland species, and generalist species, and more changed communities. Butterflies were surveyed for 5 years after the big Uljin fire in 2007. During each year, butterflies were counted monthly by the line transect method from April to October at two sites (burned vs. unburned, ~ 1.5 km routes). Specialist grassland species decreased in the year of the fire but generalist species did not increase significantly. Butterfly richness did not change but butterfly diversity decreased due to a sudden increase of a species, Polygonia c-aureum. The butterfly community in the year of the fire was different from those in later years, showing temporary change of community in the year of the fire. Species composition was significantly different between burned and unburned sites, but this phenomenon cannot be interpreted as an influence of fire due to highly variable species composition of local butterfly assemblages and the non-repetitive sampling site of the present study.     

Gender-dependent expression of pancreatic proteins in streptozotocin-induced diabetic rats

To elucidate gender-dependent protein regulation and molecular abnormalities in streptozotocin (STZ)-induced diabetes, we compared differentially expressed pancreatic proteins between male and female diabetic rats and their healthy controls using a 2-DE-based proteomic approach. In animal experiments, we found that females exposed to STZ displayed greater susceptibility towards diabetes development due to lower insulin secretion and severe β-cell damage. It was also accompanied with more impaired regulation of sex hormones, lower glucose tolerance, and higher blood glucose levels compared to male diabetic rats. Among 748 detected protein spots ranging in mass from 6 to 240 kDa between pH 3 and 10, a total of 42 proteins showed significant sexually-dimorphic regulation patterns between male and female diabetic rats. Proteomic data revealed that male and female rats displayed prominent gender-dimorphic differential regulation of pancreatic proteins involved in glycolysis, the citric acid cycle, amino acid synthesis, lipid metabolism, insulin biosynthesis, β-cell regeneration, cell signaling, as well as antioxidative and cellular stress defense. In conclusion, the current proteomic study revealed that severely impaired protein regulation in the pancreas, at least in part, is responsible for increased susceptibility of female rats to STZ-induced diabetes.     

Italian Dermestidae: notes on some species and?an?updated checklist (Coleoptera)

An up-to-date checklist of the Italian Dermestidae is provided. The presence of 95 species in Italy is confirmed, while further 5 species (Dermestes (Dermestes) vorax Motschulsky, 1860, Thorictuspilosus Peyron, 1857, T. wasmanni Reitter, 1895, Attagenus (Attagenus) simonis Reitter, 1881 and Globicornis (G.) breviclavis (Reitter, 1878)) and 1 subspecies (A. (A.) tigrinus pulcher Faldermann, 1835) are excluded from the Italian fauna.Attagenus (Attagenus) calabricus Reitter, 1881 and A. (A.) lobatus Rosenhauer, 1856 are for the first time recorded from Abruzzi and Tuscany respectively; A. (A.) silvaticus Zhantiev, 1976 is recorded for the first time from mainland Italy (Apulia); Anthrenus (Anthrenus) angustefasciatus Ganglbauer, 1904 is new to northern Italy (Friuli-Venezia Giulia), central Italy (Tuscany), Apulia and Basilicata; A. (A.) munroi Hinton, 1943 is new to central Italy (Elba Island); A. (A.) delicatus Kiesenwetter, 1851 is for the first time recorded from Apulia; Globicornis (Globicornis) fasciata (Fairmaire & Brisout de Barneville, 1859) is new to southern Italy (Basilicata); G. (Hadrotoma) sulcata (C.N.F. Brisout de Barneville, 1866) is for the first time recorded from central Italy (Abruzzi), Campania and Sicily, whileTrogoderma inclusum LeConte, 1854 is new to Apulia.Seven species (Dermestes (Dermestes) peruvianus Laporte de Castelnau, 1840, D. (Dermestinus) carnivorus Fabricius, 1775, D. (Dermestinus) hankae Háva, 1999, D. (Dermestinus) intermedius intermedius Kalík, 1951, D. (Dermestinus) szekessyi Kalík, 1950, Anthrenus (Anthrenops) coloratus Reitter, 1881 and Trogodermaangustum (Solier, 1849)) recently recorded from Italy (without further details) are discussed.The lectotype and a paralectotype are designated forAttagenus (A.) calabricus Reitter, 1881 from Calabria.Attagenus pellio (Linnaeus, 1758) var. pilosissimus Roubal, 1932 is removed from synonymy with A. (A.) pellio and recognized as a valid species (stat. prom.); it is known from Lombardy, Apulia and Calabria.     

Two newly recognized species of Hemidactylus (Squamata,Gekkonidae) from the Arabian Peninsula?and Sinai,Egypt

A recent molecular phylogeny of the Arid clade of the genus Hemidactylus revealed that the recently described H. saba and two unnamed Hemidactylus species from Sinai, Saudi Arabia and Yemen form a well-supported monophyletic group within the Arabian radiation of the genus. The name ‘Hemidactylus saba species group’ is suggested for this clade. According to the results of morphological comparisons and the molecular analyses using two mitochondrial (12S and cytb) and four nuclear (cmos, mc1r, rag1, rag2) genes, the name Hemidactylus granosus Heyden, 1827 is resurrected from the synonymy of H. turcicus for the Sinai and Saudi Arabian species. The third species of this group from Yemen is described formally as a new species H. ulii sp. n. The phylogenetic relationships of the members of ‘Hemidactylus saba species group’ are evaluated and the distribution and ecology of individual species are discussed.