A validated LC-MS/MS assay for quantification of 24(S)-hydroxycholesterol in plasma and cerebrospinal fluid

24(S)-hydroxycholesterol [24(S)-HC] is a cholesterol metabolite that is formed almost exclusively in the brain. The concentrations of 24(S)-HC in cerebrospinal fluid (CSF) and/or plasma might be a sensitive marker of altered cholesterol metabolism in the CNS. A highly sensitive 2D-LC-MS/MS assay was developed for the quantification of 24(S)-HC in human plasma and CSF. In the development of an assay for 24(S)-HC in CSF, significant nonspecific binding of 24(S)-HC was observed and resolved with the addition of 2.5% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) into CSF samples. The sample preparation consists of liquid-liquid extraction with methyl-tert-butyl ether and derivatization with nicotinic acid. Good linearity was observed in a range from 1 to 200 ng/ml and from 0.025 to 5 ng/ml, for plasma and CSF, respectively. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges. Stability of 24(S)-HC was reported under a variety of storage conditions. This method has been successfully applied to support a National Institutes of Health-sponsored clinical trial of HP-β-CD in Niemann-Pick type C1 patients, in which 24(S)-HC is used as a pharmacodynamic biomarker.     

Immobilization and stabilization of cephalosporin C acylase on aminated support by crosslinking with glutaraldehyde and further modifying with aminated macromolecules

In this work, cephalosporin C acylase (CA), a heterodimeric enzyme of industrial potential in direct hydrolysis of cephalosporin C (CPC) to 7‐aminocephalosporanic acid (7‐ACA), was covalently immobilized on the aminated support LX1000‐HA (HA) with two different protocols. The stability of CA adsorbed onto the HA support followed by crosslinking with glutaraldehyde (HA–CA–glut) was better than that of the CA covalently immobilized on the glutaraldehyde preactivated HA support (HA–glut–CA). The thermostabilization factors (compared with the free enzyme) of these two immobilized enzymes were 11.2‐fold and 2.2‐fold, respectively. In order to improve the stability of HA–CA–glut, a novel strategy based on postimmobilization modifying with aminated molecules was developed to take advantage of the glutaraldehyde moieties left on the enzyme and support. The macromolecules, such as polyethyleneimine (PEI) and chitosan, had larger effects than small molecules on the thermal stability of the immobilized enzyme perhaps due to crosslinking of the enzymes and support with each other. The quaternary structure of the CA could be much stabilized by this novel approach including physical adsorption on aminated support, glutaraldehyde treatment, and macromolecule modification. The HA–CA–glut–PEI20000 (the HA–CA–glut postmodified with PEI M_w = 20,000) had a thermostabilization factor of 20‐fold, and its substrate affinity (K_m = 14.3 mM) was better than that of HA–CA–glut (K_m = 33.4 mM). The half‐life of the immobilized enzymes HA–CA–glut–PEI20000 under the CPC‐catalyzing conditions could reach 28 cycles, a higher value than that of HA–CA–glut (21 cycles). © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:387–395, 2015     

Fullerene‐based inhibitors of HIV‐1 protease

A series of Fmoc‐Phe(4‐aza‐C₆₀)‐OH of fullerene amino acid derived peptides have been prepared by solid phase peptide synthesis, in which the terminal amino acid, Phe(4‐aza‐C₆₀)‐OH, is derived from the dipolar addition to C₆₀ of the Fmoc‐Nα‐protected azido amino acids derived from phenylalanine: Fmoc‐Phe(4‐aza‐C₆₀)‐Lys₃‐OH ( 1 ), Fmoc‐Phe(4‐aza‐C₆₀)‐Pro‐Hyp‐Lys‐OH ( 2 ), and Fmoc‐Phe(4‐aza‐C₆₀)‐Hyp‐Hyp‐Lys‐OH ( 3 ). The inhibition constant of our fullerene aspartic protease PRIs utilized FRET‐based assay to evaluate the enzyme kinetics of HIV‐1 PR at various concentrations of inhibitors. Simulation of the docking of the peptide Fmoc‐Phe‐Pro‐Hyp‐Lys‐OH overestimated the inhibition, while the amino acid PRIs were well estimated. The experimental results show that C₆₀‐based amino acids are a good base structure in the design of protease inhibitors and that their inhibition can be improved upon by the addition of designer peptide sequences. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Modulation of adenosine A1 and A2A receptors in C6 glioma cells during hypoxia: involvement of endogenous adenosine

During hypoxia, extracellular adenosine levels are increased to prevent cell damage, playing a neuroprotective role mainly through adenosine A₁ receptors. The aim of the present study was to analyze the effect of hypoxia in both adenosine A₁ and A_2A receptors endogenously expressed in C6 glioma cells. Two hours of hypoxia (5% O₂) caused a significant decrease in adenosine A₁ receptors. The same effect was observed at 6 h and 24 h of hypoxia. However, adenosine A_2A receptors were significantly increased at the same times. These effects were not due to hypoxia-induced alterations in cells number or viability. Changes in receptor density were not associated with variations in the rate of gene expression. Furthermore, hypoxia did not alter HIF-1α expression in C6 cells. However, HIF-3α, CREB and CREM were decreased. Adenosine A₁ and A_2A receptor density in normoxic C6 cells treated with adenosine for 2, 6 and 24 h was similar to that observed in cells after oxygen deprivation. When C6 cells were subjected to hypoxia in the presence of adenosine deaminase, the density of receptors was not significantly modulated. Moreover, DPCPX, an A₁ receptor antagonist, blocked the effects of hypoxia on these receptors, while ZM241385, an A_2A receptor antagonist, was unable to prevent these changes. These results suggest that moderate hypoxia modulates adenosine receptors and cAMP response elements in glial cells, through a mechanism in which endogenous adenosine and tonic A₁ receptor activation is involved.     

Dancing with the sterols: Critical roles for ABCG1, ABCA1, miRNAs, and nuclear and cell surface receptors in controlling cellular sterol homeostasis

ATP binding cassette (ABC) transporters represent a large and diverse family of proteins that transport specific substrates across a membrane. The importance of these transporters is illustrated by the finding that inactivating mutations within 17 different family members are known to lead to specific human diseases. Clinical data from humans and/or studies with mice lacking functional transporters indicate that ABCA1, ABCG1, ABCG4, ABCG5 and ABCG8 are involved in cholesterol and/or phospholipid transport. This review discusses the multiple mechanisms that control cellular sterol homeostasis, including the roles of microRNAs, nuclear and cell surface receptors and ABC transporters, with particular emphasis on recent findings that have provided insights into the role(s) of ABCG1. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Mechanisms of Glycine Release in Mouse Brain Stem Slices

In the brain stem glycine is associated with multiple sensory and visceral regulations, being involved in, for instance, cardiovascular, respiratory and auditory functions. We here studied the mechanisms of the release of preloaded [³H]glycine from mouse brain stem slices in a superfusion system. A depolarizing concentration of K⁺ ions (50 mM) evoked glycine release, but in the absence of Ca²⁺ the effect was attenuated, indicating that a part of the evoked release represents Ca²⁺-dependent exocytosis. The Ca²⁺-independent release was enhanced by omission of Na⁺ and Cl⁻. The stimulatory effect of extracellular glycine confirmed the involvement of transporters functioning in a reverse direction. A part of the release is mediated by Na⁺ and Cl⁻ channels, since it was inhibited by the inhibitors of these, riluzole and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonate, respectively. Glycine release was potentiated by the activation of protein kinase C and diminished by increasing cyclic guanosine monophosphate levels with a phosphodiesterase inhibitor, zaprinast. The release was also modulated by the phospholipase inhibitor quinacrine and the tyrosine kinase inhibitor genistein. Adenosine A₁ receptors likewise regulate glycine release, since it was enhanced by their agonist R(−)N⁶-(2-phenylisopropyl)adenosine, which effect was blocked by the antagonist 8-cyclopentyl-1,3-dipropylxanthine. The ionotropic glutamate receptor agonists N-methyl-d-aspartate, kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate failed to have any effects contrary to their effects in higher brain regions, e.g., in the hippocampus. The group I and III metabotropic glutamate receptor agonists (S)-3,5-dihydroxyphenylglycine and O-phospho-l-serine, respectively, increased the release in a receptor-mediated manner. Glycine release in the brain stem was also markedly enhanced by cell-damaging conditions, including hypoxia, hypoglycemia and ischemia.     

Effects of oxidative stress on phospholipid signaling in rat cultured astrocytes and brain slices

Although reactive oxygen species (ROS) are conventionally viewed as toxic by-products of cellular metabolism, a growing body of evidence suggests that they may act as signaling molecules. We have studied the effects of hydrogen peroxide (H(2)O(2))-induced oxidative stress on phospholipid signaling in cultured rat cortical astrocytes. H(2)O(2) stimulated the formation of phosphatidic acid and the accumulation of phosphatidylbutanol, a product of the phospholipase D (PLD)-catalyzed transphosphatidylation reaction. The effect of exogenous H(2)O(2) on the PLD response was mimicked by menadione-induced production of endogenous H(2)O(2). Oxidative stress also elicited inositol phosphate accumulation resulting from phosphoinositide phospholipase C (PLC) activation. The PLD response to H(2)O(2) was totally suppressed by chelation of both extracellular and cytosolic Ca(2+) with EGTA and BAPTA/AM, respectively. Furthermore, H(2)O(2)-induced PLD stimulation was completely abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide and chelerythrine and by PKC down-regulation. Activation of PLD by H(2)O(2) was also inhibited by the protein-tyrosine kinase inhibitor genistein. Finally, H(2)O(2) also stimulated both PLC and PLD in rat brain cortical slices. These results show for the first time that oxidative stress elicits phospholipid breakdown by both PLC and PLD in rat cultured astrocytes and brain slices.     

枝孢属4个吉林省新记录种

记录枝孢属(Cladosporium Link)4个吉林省新记录种:连翘枝孢(C. forsytiae Z.Y.Zhanget T.Zhang)、三宅枝孢(C.miyakei Saccardoet Trotter)、柳枝孢(C.salicis Moseszet Smarods)、黑星状枝孢(C.venturicides Saccardo)。文中对这些种分别进行了描述,并附有线条图。标本保存于吉林农业大学菌物标本室(HMJAU)。