Interleukin-1-induced neurotoxicity is mediated by glia and requires caspase activation and free radical release

Interleukin (IL)-1 expression is induced rapidly in response to diverse CNS insults and is a key mediator of experimentally induced neuronal injury. However, the mechanisms of IL-1-induced neurotoxicity are unknown. The aim of the present study was to examine the toxic effects of IL-1 on rat cortical cell cultures. Treatment with IL-1beta did not affect the viability of pure cortical neurones. However, IL-1 treatment of cocultures of neurones with glia or purified astrocytes induced caspase activation resulting in neuronal death. Neuronal cell death induced by IL-1 was prevented by pre-treatment with the IL-1 receptor antagonist, the broad spectrum caspase inhibitor Boc-Asp-(OMe)-CH(2)F or the antioxidant alpha-tocopherol. The NMDA receptor antagonist dizolcipine (MK-801) attenuated cell death induced by low doses of IL-1beta but the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) had no effect. Inhibition of inducible nitric oxide synthase with N(omega)-nitro-l-arginine methyl ester had no effect on neuronal cell death induced by IL-1beta. Thus, IL-1 activates the IL-1 type 1 receptor in astrocytes to induce caspase-dependent neuronal death, which is dependent on the release of free radicals and may contribute to neuronal cell death in CNS diseases.     

Asymmetric recruitment of dynein to spindle poles and microtubules promotes proper spindle orientation in yeast

The orientation of the mitotic spindle plays a key role in determining whether a polarized cell will divide symmetrically or asymmetrically. In most cell types, cytoplasmic dynein plays a critical role in spindle orientation. However, how dynein directs opposite spindle poles toward distinct and predetermined cell ends is poorly understood. Here, we show that dynein distributes preferentially to the spindle pole bodies (SPB) and astral microtubules (MTs) proximal to the bud in metaphase yeast cells. Dynein asymmetry depended on the bud neck kinases Elm1, Hsl1, and Gin4, on the spindle pole components Cnm67 and Cdk1, and on the B-type cyclins Clb1 and Clb2. Furthermore, phenotypic and genetic studies both indicated that dynein is unable to orient the spindle when it localizes to both poles and associated microtubules. Together, our data indicate that proper orientation of the spindle requires dynein to act on a single spindle pole.     

Erythrocyte and plasma protein modification in alcoholism: a possible role of acetaldehyde

Analysis of the oxidative modification of plasma and erythrocyte ghost proteins of chronic alcoholic subjects and healthy non-alcoholics has been performed. It was found that increased levels of protein carbonyls in both plasma and erythrocyte ghosts from alcoholic subjects occurred in comparison to the levels found in preparations from non-alcoholics. Plasma proteins from alcoholic subjects did not show evidence of cross-linking, although plasma protein concentration and composition were changed. In alcoholic subjects who displayed no evidence of abnormal erythrocyte morphology no cross-linking of erythrocyte ghost proteins was detectable, whereas the ghosts obtained from alcoholic subjects who displayed morphologically abnormal erythrocytes contained cross-linked proteins. The in vitro treatment with acetaldehyde of erythrocytes from non-alcoholics caused increased levels of protein carbonyls and cross-linking products in erythrocyte ghost preparations which were similar to those found in severe alcoholics. It is concluded that chronic alcohol consumption can cause abnormal erythrocyte morphology and increased erythrocyte fragility as a result of oxidation and cross-linking of erythrocyte ghost proteins. These effects can be ascribed, in part, to exposure of erythrocytes to circulatory acetaldehyde which is a product of ethanol metabolism.     

Online immunoaffinity liquid chromatography/tandem mass spectrometry determination of a type II collagen peptide biomarker in rat urine: Investigation of the impact of collision-induced dissociation fluctuation on peptide quantitation

Proteolytic fragments of type II collagen, a major component of joint tissue, have recently been identified as biomarkers for osteoarthritis, a progressive disease associated with cartilage degeneration. A liquid chromatography/tandem mass spectrometry (MS/MS) assay that utilizes online immunoaffinity chromatography and column switching was developed in our laboratory for the neoepitope of type II collagen (NET2C). During method development, peptide collision-induced dissociation (CID) was found to be a significant source of assay variation, which exceeded 10% CV, despite the fact that a stable-isotope-labeled (SIL) internal standard was used to minimize imprecision. This phenomenon was studied in detail using peptides and associated SIL internal standards of varying lengths and amino acid compositions. Variability in peptide CID necessitated the monitoring of multiple MS/MS transitions to obtain acceptable assay precision. The assay was subsequently validated to measure NET2C concentrations in rat urine over the range of 0.1 to 10 ng/mL. The interday accuracy and precision ranged from 3.9 to 13.1 (%CV) and 10.7 to 5.3 (%RE), respectively, across the range of validated concentrations. A specific application of the assay is presented in which the role of estrogen deficiency in the development and progression of osteoarthritis was investigated. In this study, the effect of estrogen on lowering NET2C concentrations in urine in ovariectomized rats was demonstrated.     

Helicobacter pylori-induced downregulation of the secretory leukocyte protease inhibitor (SLPI) in gastric epithelial cell lines and its functional relevance for H. pylori-mediated diseases

The secretory leukocyte protease inhibitor (SLPI) exerts antiproteolytic activity towards serine proteases, as well as anti-microbial and anti-inflammatory effects. To investigate its role in H. pylori-mediated diseases, SLPI expression was analyzed by RT-PCR, ELISA and immunohistochemistry in clinical samples and gastric tumor cell lines. Determination of the mucosal SLPI levels in 126 patients confirmed the previously reported downregulation of SLPI in H. pylori-infected patients. The lower SLPI levels in antral biopsies of H. pylori-positive subjects were associated with a 30-fold increase (p<0.01) in neutrophil elastase activity, and a significant negative correlation was demonstrated for both parameters (R=-0.63, p=0.0002). Eradication of the bacterium in a long-term study (5-7 years) led to a recovery of mucosal SLPI expression. In vitro experiments using four gastric tumor cell lines (AGS, MKN-28, MKN-45, NCI-N87) generally confirmed the clinical findings. While the co-incubation of these cell lines with H. pylori resulted in lower or unchanged SLPI protein levels, the corresponding SLPI mRNA amounts were upregulated by up to five-fold (p=0.006) in all cell lines. Taken together, these results indicate that the reduction in antral SLPI levels in H. pylori-infected subjects has a functional relevance for gastric mucosa and the H. pylori-induced decrease in SLPI is primarily regulated at the posttranslational level.     

Structural and biophysical characterization of the EphB4*ephrinB2 protein-protein interaction and receptor specificity

Increasing evidence implicates the interaction of the EphB4 receptor with its preferred ligand, ephrinB2, in pathological forms of angiogenesis and in tumorigenesis. To identify the molecular determinants of the unique specificity of EphB4 for ephrinB2, we determined the crystal structure of the ligand binding domain of EphB4 in complex with the extracellular domain of ephrinB2. This structural analysis suggested that one amino acid, Leu-95, plays a particularly important role in defining the structural features that confer the ligand selectivity of EphB4. Indeed, all other Eph receptors, which promiscuously bind many ephrins, have a conserved arginine at the position corresponding to Leu-95 of EphB4. We have also found that amino acid changes in the EphB4 ligand binding cavity, designed based on comparison with the crystal structure of the more promiscuous EphB2 receptor, yield EphB4 variants with altered binding affinity for ephrinB2 and an antagonistic peptide. Isothermal titration calorimetry experiments with an EphB4 Leu-95 to arginine mutant confirmed the importance of this amino acid in conferring high affinity binding to both ephrinB2 and the antagonistic peptide ligand. Isothermal titration calorimetry measurements also revealed an interesting thermodynamic discrepancy between ephrinB2 binding, which is an entropically driven process, and peptide binding, which is an enthalpically driven process. These results provide critical information on the EphB4*ephrinB2 protein interfaces and their mode of interaction, which will facilitate development of small molecule compounds inhibiting the EphB4*ephrinB2 interaction as novel cancer therapeutics.     

Mutation in a "tesB-like" hydroxyacyl-coenzyme A-specific thioesterase gene causes hyperproduction of extracellular polyhydroxyalkanoates by Alcanivorax borkumensis SK2

A novel mutant of the marine oil-degrading bacterium Alcanivorax borkumensis SK2, containing a mini-Tn5 transposon disrupting a "tesB-like" acyl-coenzyme A (CoA) thioesterase gene, was found to hyperproduce polyhydroxyalkanoates (PHA), resulting in the extracellular deposition of this biotechnologically important polymer when grown on alkanes. The tesB-like gene encodes a distinct novel enzyme activity, which acts exclusively on hydroxylated acyl-CoAs and thus represents a hydroxyacyl-CoA-specific thioesterase. Inactivation of this enzyme results in the rechanneling of CoA-activated hydroxylated fatty acids, the cellular intermediates of alkane degradation, towards PHA production. These findings may open up new avenues for the development of simplified biotechnological processes for the production of PHA as a raw material for the production of bioplastics.     

Mouse Emi2 is required to enter meiosis II by reestablishing cyclin B1 during interkinesis

During interkinesis, a metaphase II (MetII) spindle is built immediately after the completion of meiosis I. Oocytes then remain MetII arrested until fertilization. In mouse, we find that early mitotic inhibitor 2 (Emi2), which is an anaphase-promoting complex inhibitor, is involved in both the establishment and the maintenance of MetII arrest. In MetII oocytes, Emi2 needs to be degraded for oocytes to exit meiosis, and such degradation, as visualized by fluorescent protein tagging, occurred tens of minutes ahead of cyclin B1. Emi2 antisense morpholino knockdown during oocyte maturation did not affect polar body (PB) extrusion. However, in interkinesis the central spindle microtubules from meiosis I persisted for a short time, and a MetII spindle failed to assemble. The chromatin in the oocyte quickly decondensed and a nucleus formed. All of these effects were caused by the essential role of Emi2 in stabilizing cyclin B1 after the first PB extrusion because in Emi2 knockdown oocytes a MetII spindle was recovered by Emi2 rescue or by expression of nondegradable cyclin B1 after meiosis I.     

Fragrep： An Efficient Search Tool for Fragmented Patterns in Genomic Sequences

Many classes of non-coding RNAs （ncRNAs; including Y RNAs, vault RNAs, RNase P RNAs, and MRP RNAs, as well as a novel class recently discovered in Dictyostelium discoideum） can be characterized by a pattern of short but well-conserved sequence elements that are separated by poorly conserved regions of sometimes highly variable lengths. Local alignment algorithms such as BLAST are therefore ill-suited for the discovery of new homologs of such ncRNAs in genomic sequences. The Fragrep tool instead implements an efficient algorithm for detecting the pattern fragments that occur in a given order. For each pattern fragment, the mismatch tolerance and bounds on the length of the intervening sequences can be specified separately. Furthermore, matches can be ranked by a statistically well-motivated scoring scheme.