Novel substituted 4-phenyl-[1,3]dioxanes: potent and selective orexin receptor 2 (OX(2)R) antagonists

Orexins, also termed hypocretins, consist of two neuropeptide agonists (orexin A and B) interacting with two known G-protein coupled receptors (OX(1)R and OX(2)R). In addition to other biological functions, the orexin-2 receptor is thought to be an important modulator of sleep and wakefulness. Herein we describe a series of novel, selective OX(2)R antagonists consisting of substituted 4-phenyl-[1,3]dioxanes. One such antagonist is compound 9, 1-(2,4-dibromo-phenyl)-3-((4S,5S)-2,2-dimethyl-4-phenyl-[1,3]dioxan-5-yl)-urea, which is bound by the OX(2)R with a pK(i) of 8.3, has a pK(b) of 7.9, and is 600-fold selective for the OX(2)R over the OX(1)R.     

Mitochondrial perturbation attenuates bile acid-induced cytotoxicity

Hydrophobic bile acids such as deoxycholate (DOC) are known to damage liver cells during cholestasis and promote colon cancer. Cellular stresses induced by bile acids, which include mitochondrial and endoplasmic reticulum (ER) stresses, can result in apoptosis. We found that inhibition of mitochondrial complexes I–V with rotenone, thenoyltrifluoroacetone (TTFA), antimycin A, myxothiazol or oligomycin strongly protected against DOC-induced apoptosis of HCT-116 cells. To understand the mechanism of this protection, we explored the ability of these specific inhibitors to reduce DOC-induced mitochondrial and ER stresses. Different inhibitors markedly reduced DOC-induction of mitochondrial condensation, the DOC-induced decrease in mitochondrial membrane potential and the DOC-induced dilatation of the ER (evidence of ER stress). A dramatic induction of nucleolar segregation by antimycin A and myxothiazol, two distinct complex III inhibitors, was also observed. These findings strongly implicate mitochondrial crosstalk with apoptotic signaling pathways and mitochondrial–nucleolar crosstalk in the development of apoptosis resistance in the colon.     

Intragranular vesiculotubular compartments are involved in piecemeal degranulation by activated human eosinophils

Eosinophils, leukocytes involved in allergic, inflammatory and immunoregulatory responses, have a distinct capacity to rapidly secrete preformed granule-stored proteins through piecemeal degranulation (PMD), a secretion process based on vesicular transport of proteins from within granules for extracellular release. Eosinophil-specific granules contain cytokines and cationic proteins, such as major basic protein (MBP). We evaluated structural mechanisms responsible for mobilizing proteins from within eosinophil granules. Human eosinophils stimulated for 30-60 min with eotaxin, regulated on activation, normal, T-cell expressed and secreted (RANTES) or platelet activating factor exhibited ultrastructural features of PMD (e.g. losses of granule contents) and extensive vesiculotubular networks within emptying granules. Brefeldin A inhibited granule emptying and collapsed intragranular vesiculotubular networks. By immunonanogold ultrastructural labelings, CD63, a tetraspanin membrane protein, was localized within granules and on vesicles outside of granules, and mobilization of MBP into vesicles within and extending from granules was demonstrated. Electron tomography with three dimension reconstructions revealed granule internal membranes to constitute an elaborate tubular network able to sequester and relocate granule products upon stimulation. We provide new insights into PMD and identify eosinophil specific granules as organelles whose internal tubulovesicular networks are important for the capacity of eosinophils to secrete, by vesicular transport, their content of preformed and granule-stored cytokines and cationic proteins.     

Sequence polymorphism in polyploid wheat and their d-genome diploid ancestor

Sequencing was used to investigate the origin of the D genome of the allopolyploid species Triticum aestivum and Aegilops cylindrica. A 247-bp region of the wheat D-genome Xwye838 locus, encoding ADP-glucopyrophosphorylase, and a 326-bp region of the wheat D-genome Gss locus, encoding granule-bound starch synthase, were sequenced in a total 564 lines of hexaploid wheat (T. aestivum, genome AABBDD) involving all its subspecies and 203 lines of Aegilops tauschii, the diploid source of the wheat D genome. In Ae. tauschii, two SNP variants were detected at the Xwye838 locus and 11 haplotypes at the Gss locus. Two haplotypes with contrasting frequencies were found at each locus in wheat. Both wheat Xwye838 variants, but only one of the Gss haplotypes seen in wheat, were found among the Ae. tauschii lines. The other wheat Gss haplotype was not found in either Ae. tauschii or 70 lines of tetraploid Ae. cylindrica (genomes CCDD), which is known to hybridize with wheat. It is concluded that both T. aestivum and Ae. cylindrica originated recurrently, with at least two genetically distinct progenitors contributing to the formation of the D genome in both species.     

Identification of bilirubin reduction products formed by Clostridium perfringens isolated from human neonatal fecal flora

Urobilinoids belong to the heterogenous group of degradation products of bilirubin formed in the gastrointestinal tract by intestinal microflora. Among them urobilinogen and stercobilinogen with their respective oxidation products, urobilin and stercobilin, are the most important compounds. The aim of present study was to analyze the products of bacterial reduction of bilirubin in more detail. The strain of Clostridium perfringens isolated from neonatal stools, capable of reducing bilirubin, was used in the study. Bacteria were incubated under anaerobic conditions with various native as well as synthetic bile pigments, including radiolabeled unconjugated bilirubin (UCB). Their reduction products were extracted from media and separated following thin layer chromatography. Pigments isolated were analyzed by spectrophotometry, spectrofluorometry and mass spectrometry. In a special set of experiments, bilirubin diglucuronide was incubated with either bacterial lysate or partially purified bilirubin reductase and beta-glucuronidase to reveal whether bilirubin glucuronides may be directly reduced onto conjugated urobilinoids. A broad substrate activity was detected in the investigated strain of C. perfringens and a series of bilirubin reduction products was identified. These products were separated in the form of their respective chromogens and further oxidized. Based on their physical-chemical properties, as well as mass spectra, end-catabolic bilirubin products were identified to belong to urobilinogen species. The reduction process, catalyzed enzymatically by the studied bacterial strain, does not proceed to stercobilinogen. Bilirubin diglucuronide is not reduced onto urobilinoid conjugates, glucuronide hydrolysis must precede double bond reduction and thus UCB is reduced much faster.     

Regulation of microtubule formation in activated mast cells by complexes of gamma-tubulin with Fyn and Syk kinases

Aggregation of the high-affinity IgE receptors (FcepsilonRIs) on the surface of granulated mast cells initiates a chain of signaling events culminating in the release of allergy mediators. Although microtubules are involved in mast cell degranulation, the molecular mechanism that controls microtubule rearrangement after FcepsilonRI triggering is poorly understood. In this study, we show that the activation of bone marrow-derived mast cells (BMMCs) induced by FcepsilonRI aggregation or treatment with pervanadate leads to a rapid polymerization of microtubules. This polymerization was not dependent on the presence of Lyn kinase as determined by experiments with BMMCs isolated from Lyn-negative mice. One of the key regulators of microtubule polymerization is gamma-tubulin. Immunoprecipitation experiments revealed that gamma-tubulin from activated cells formed complexes with Fyn and Syk protein tyrosine kinases and several tyrosine phosphorylated proteins from both wild-type and Lyn(-/-) BMMCs. Pretreatment of the cells with Src-family or Syk-family selective tyrosine kinase inhibitors, PP2 or piceatannol, respectively, inhibited the formation of microtubules and reduced the amount of tyrosine phosphorylated proteins in gamma-tubulin complexes, suggesting that Src and Syk family kinases are involved in the initial stages of microtubule formation. This notion was corroborated by pull-down experiments in which gamma-tubulin complex bounds to the recombinant Src homology 2 and Src homology 3 domains of Fyn kinase. We propose that Fyn and Syk kinases are involved in the regulation of binding properties of gamma-tubulin and/or its associated proteins, and thus modulate the microtubule nucleation in activated mast cells.     

Synthesis and biological activity of olomoucine II

Based on our previous experiences with synthesis of purines, novel 2,6,9-trisubstituted purine derivatives were prepared and assayed for the ability to inhibit CDK1/cyclin B kinase. One of newly synthesized compounds designated as olomoucine II, 6-[(2-hydroxybenzyl)amino]-2-[[1-(hydroxymethyl)propyl]amino]-9-isopropylpurine, displays 10 times higher inhibitory activity than roscovitine, potent and specific CDK1 inhibitor. Olomoucine II in vitro cytotoxic activity exceeds purvalanol A, the most potent CDK inhibitor, as it kills the CEM cells with IC(50) value of 3.0 microM.     

Pomegranate seed oil reduces intestinal damage in a rat model of necrotizing enterocolitis

Pomegranate seed oil (PSO), which is the major source of conjugated linolenic acids such as punicic acid (PuA), exhibits strong anti-inflammatory properties. Necrotizing enterocolitis (NEC) is a devastating disease associated with severe and excessive intestinal inflammation. The aim of this study was to evaluate the effects of orally administered PSO on the development of NEC, intestinal epithelial proliferation, and cytokine regulation in a rat model of NEC. Premature rats were divided into three groups: dam fed (DF), formula-fed rats (FF), or rats fed with formula supplemented with 1.5% of PSO (FF + PSO). All groups were exposed to asphyxia/cold stress to induce NEC. Intestinal injury, epithelial cell proliferation, cytokine production, and trefoil factor 3 (Tff3) production were evaluated in the terminal ileum. Oral administration of PSO (FF+PSO) decreased the incidence of NEC from 61 to 26%. Feeding formula with PSO improved enterocyte proliferation in the site of injury. Increased levels of proinflammatory IL-6, IL-8, IL-12, IL-23, and TNF-α in the ileum of FF rats were normalized in PSO-treated animals. Tff3 production in the FF rats was reduced compared with DF but not further affected by the PSO. In conclusion, administration of PSO protects against NEC in the neonatal rat model. This protective effect is associated with an improvement of intestinal epithelial homeostasis and a strong anti-inflammatory effect of PSO on the developing intestinal mucosa.     

Dynein,Lis1 and CLIP‐170 counteract Eg5‐dependent centrosome separation during bipolar spindle assembly

Bipolar spindle assembly critically depends on the microtubule plus‐end‐directed motor Eg5 that binds antiparallel microtubules and slides them in opposite directions. As such, Eg5 can produce the necessary outward force within the spindle that drives centrosome separation and inhibition of this antiparallel sliding activity results in the formation of monopolar spindles. Here, we show that upon depletion of the minus‐end‐directed motor dynein, or the dynein‐binding protein Lis1, bipolar spindles can form in human cells with substantially less Eg5 activity, suggesting that dynein and Lis1 produce an inward force that counteracts the Eg5‐dependent outward force. Interestingly, we also observe restoration of spindle bipolarity upon depletion of the microtubule plus‐end‐tracking protein CLIP‐170. This function of CLIP‐170 in spindle bipolarity seems to be mediated through its interaction with dynein, as loss of CLIP‐115, a highly homologous protein that lacks the dynein–dynactin interaction domain, does not restore spindle bipolarity. Taken together, these results suggest that complexes of dynein, Lis1 and CLIP‐170 crosslink and slide microtubules within the spindle, thereby producing an inward force that pulls centrosomes together.     

Differential expression of thymosin beta-10 by early passage and senescent vascular endothelium is modulated by VPF/VEGF: evidence for senescent endothelial cells in vivo at sites of atherosclerosis.

VPF/VEGF acts selectively on the vascular endothelium to enhance permeability, induce cell migration and division, and delay replicative senescence. To understand the changes in gene expression during endothelial senescence, we investigated genes that were differentially expressed in early vs. late passage (senescent) human dermal endothelial cells (HDMEC) using cDNA array hybridization. Early passage HDMEC cultured with or without VPF/VEGF overexpressed 9 and underexpressed 6 genes in comparison with their senescent counterparts. Thymosin beta-10 expression was modulated by VPF/VEGF and was strikingly down-regulated in senescent EC. The beta-thymosins are actin G-sequestering peptides that regulate actin dynamics and are overexpressed in neoplastic transformation. We have also identified senescent EC in the human aorta at sites overlying atherosclerotic plaques. These EC expressed senescence-associated neutral beta-galactosidase and, in contrast to adventitial microvessel endothelium, exhibited weak staining for thymosin beta-10. ISH performed on human malignant tumors revealed strong thymosin beta-10 expression in tumor blood vessels. This is the first report that Tbeta-10 expression is significantly reduced in senescent EC, that VPF/VEGF modulates thymosin beta-10 expression, and that EC can become senescent in vivo. The reduced expression of thymosin beta-10 may contribute to the senescent phenotype by reducing EC plasticity and thus impairing their response to migratory stimuli.