Effects of corticosteroid-induced apoptosis on airway epithelial wound closure in vitro

Damage to the airway epithelium is common in asthma. Corticosteroids induce apoptosis in and suppress proliferation of airway epithelial cells in culture. Whether apoptosis contributes to impaired epithelial cell repair after injury is not known. We examined whether corticosteroids would impair epithelial cell migration in an in vitro model of wound closure. Wounds (approximately 0.5-1.3 mm2) were created in cultured 1HAEo- human airway epithelial cell monolayers, after which cells were treated with up to 10 microM dexamethasone or budesonide for 24 h. Cultured cells were pretreated for 24 or 48 h with dexamethasone to observe the effect of long-term exposure on wound closure. After 12 h, the remaining wound area in monolayers pretreated for 48 h with 10 microM dexamethasone was 43+/-18% vs. 10+/-8% for untreated control monolayers. The addition of either corticosteroid immediately after injury did not slow closure significantly. After 12 h the remaining wound area in monolayers treated with 10 microM budesonide was 39+/-4% vs. 43+/-3% for untreated control monolayers. The proportion of apoptotic epithelial cells as measured by terminal deoxynucleotidyltransferase-mediated dUTP biotin nick end labeling both at and away from the wound edge was higher in monolayers treated with budesonide compared with controls. However, wound closure in the apoptosis-resistant 1HAEo-.Bcl-2+ cell line was not different after dexamethasone treatment. We demonstrate that corticosteroid treatment before mechanical wounding impairs airway epithelial cell migration. The addition of corticosteroids after injury does not slow migration, despite their ability to induce apoptosis in these cells.     

β-Amyloid enhances intracellular calcium rises mediated by repeated activation of intracellular calcium stores and nicotinic receptors in acutely dissociated rat basal forebrain neurons

β-Amyloid, a 39–43 amino acid peptide, may exert its biological effects via neuronal nicotinic acetylcholine receptors. Using the ratiometric dye, fura-2, we examined the effect of soluble β-amyloid_1–42 on the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) in acutely dissociated rat basal forebrain neurons. Focal applications of nicotine (0.5–20 mM), evoked dose-dependent increases in intracellular [Ca²⁺]_i that were mediated by the entry of extracellular Ca²⁺ via nicotinic acetylcholine receptors, and the release of intracellular Ca²⁺ from stores. With repeated nicotine challenges, the nicotinic responses were potentiated by 98 ± 12% (P < 0.05) while β-amyloid_1–42 (100 nM) was present for ∼5 min. This potentiation became larger during the subsequent washout of β-amyloid_1–42, which was associated with a gradual rise in baseline [Ca²⁺]_i. Application of β-amyloid_1–42 by itself did not alter [Ca²⁺]_i, and β-amyloid_1–42 also had no significant effect on the response to repeated KCl challenges. Therefore, β-amyloid_1–42 caused neither gross disturbance of cellular Ca²⁺ homeostasis nor enhancement of voltage-gated Ca²⁺ channels. Interestingly, β-amyloid_1–42 transiently potentiated the response to repeated caffeine challenges, which was also associated with a transient rise in baseline [Ca²⁺]_i. β-amyloid_1–42 potentiation of nicotine-evoked rises in [Ca²⁺]_i was reversed by the SERCA pump inhibitor, thapsigargin, and the mitochondrial Na⁺/Ca²⁺ exchanger inhibitor, CGP-37157. These results suggest that the dysregulation of [Ca²⁺]_i by β-amyloid_1–42 during multiple challenges with nicotine or caffeine involved the sensitization or overfilling of intracellular stores that are maintained by SERCA pump and Ca²⁺ efflux from the mitochondria.     

Resveratrol derivative,trans‐3,5,4′‐trimethoxystilbene,exerts antiangiogenic and vascular‐disrupting effects in zebrafish through the downregulation of VEGFR2 and cell‐cycle modulation

Angiogenesis plays an important role in the development of neoplastic diseases such as cancer. Resveratrol and its derivatives exert antiangiogenic effects, but the mechanisms of their actions remain unclear. The aim of this study was to evaluate the antiangiogenic activity of resveratrol and its derivative trans‐3,5,4′‐trimethoxystilbene in vitro using human umbilical vein endothelial cells (HUVECs) and in vivo using transgenic zebrafish, and to clarify their mechanisms of action in zebrafish by gene expression analysis of the vascular endothelial growth factor (VEGF) receptor (VEGFR2/KDR) and cell‐cycle analysis. trans‐3,5,4′‐Trimethoxystilbene showed significantly more potent antiangiogenic activity than that of resveratrol in both assays. In zebrafish, trans‐3,5,4′‐trimethoxystilbene caused intersegmental vessel regression and downregulated VEGFR2 mRNA expression. Trans‐3,5,4′‐trimethoxystilbene also induced G2/M cell‐cycle arrest, most specifically in endothelial cells of zebrafish embryos. We propose that the antiangiogenic and vascular‐targeting activities of trans‐3,5,4′‐trimethoxystilbene result from the downregulation of VEGFR2 expression and cell‐cycle arrest at G2/M phase. J. Cell. Biochem. 109: 339–346, 2010. © 2009 Wiley‐Liss, Inc.     

Complete Genome Sequence of Staphylococcus lugdunensis Strain HKU09-01

Staphylococcus lugdunensis is a member of the coagulase-negative staphylococci and commonly found as part of the human skin flora. It is a significant cause of catheter-related bacteremia and also causes serious infections like native valve endocarditis in previously healthy individuals. We report the complete genome sequence of this medically important bacterium.Staphylococcus lugdunensis is a member of the coagulase-negative staphylococci (CoNS) commonly colonizing the human skin and mucosal membranes. While the genus Staphylococcus contains 48 named species currently, only a few species, notably S. aureus, are coagulase positive. Thus, the phenotypic characteristic is routinely tested in the medical microbiological laboratory for rapid differentiation of the highly pathogenic S. aureus from the other staphylococci. Among the CoNS, only a few species are known to cause human disease, usually in the form of opportunistic infections only (6). However, S. lugdunensis is an important exception (3). Besides causing catheter-related bacteremia similar to other CoNS, it causes a variety of severe nosocomial and community-acquired infections, including native valve endocarditis, a devastating and potentially fatal disease that can affect previously healthy individuals. Another unusual feature are the susceptibilities of S. lugdunensis isolates to multiple antimicrobial agents even when the incidence of multiple-drug-resistant CoNS and S. aureus occurrences are increasing in both hospital and community settings (4, 5).The genome sequence of S. lugdunensis strain HKU09-01 was determined by high-throughput sequencing performed on a GS FLX system (Roche Diagnostics, Basel, Switzerland), with approximately 45-fold coverage of the genome. This clinical strain was previously isolated from the culture of pus from a skin swab. Genome assembly was performed using the Newbler assembler, resulting in 30 large contigs (>500 bp in size). The contigs were then ordered and oriented into one scaffold using OSLay (11). The genome-finishing strategy for S. lugdunensis was similar to that employed for our previously sequenced Laribacter hongkongensis genome (12). Briefly, gap closures were performed by genomic PCR followed by DNA sequencing of amplification products on an ABI 3130xl sequencer (Applied Biosystems, CA). The finished sequence was validated by genome macrorestriction analysis using multiple rare-cutting enzymes and visualization by pulsed-field gel electrophoresis. Protein coding regions were predicted with Glimmer3 (2), and automatic genome annotation was performed on the RAST server (1). Additionally, annotation of tRNA and transfer-messenger RNA (tmRNA) genes was performed using tRNAScan-SE (10) and ARAGORN (9). Identification of rRNA genes was performed using RNAmmer (8).The genome of S. lugdunensis strain HKU09-01 consists of a circular 2,658,366-bp chromosome with G+C content of 33.87%, similar to those of other staphylococci. No plasmids are present in the sequenced strain. The genome contains 61 tRNA genes for all amino acids and 2,489 predicted protein-coding genes. Eight putative genomic islands were identified, and one actually consists of a pair of duplicated 32-kb genomic regions. Similar to Staphylococcus saprophyticus (7), but different from the other staphylococci, the genome contains 6 rRNA operons, one of them having the unusual organization 5S-16S-23S-5S.With the availability of the present genome sequence, S. lugdunensis now joins other staphylococcal species with human pathogenic potential, like S. aureus, S. epidermidis, S. haemolyticus, and S. saprophyticus, to have at least one reference genome available. Further in-depth analysis will be necessary to fully elucidate the genomic differences that may explain the variation in virulence of the staphylococcal species.     

Biotin synthase mechanism: mutagenesis of the YNHNLD conserved motif

Biotin synthase, a member of the "radical SAM" family, catalyzes the final step of the biotin biosynthetic pathway, namely, the insertion of a sulfur atom into dethiobiotin (DTB). The active form of the enzyme contains two iron-sulfur clusters, a [4Fe-4S](2+) cluster liganded by Cys-53, Cys-57, and Cys-60 and the S-adenosylmethionine (AdoMet or SAM) cosubstrate and a [2Fe-2S](2+) cluster liganded by Cys-97, Cys-128, Cys-188, and Arg-260. Single-point mutation of each of these six conserved cysteines produced inactive variants. In this work, mutants of other highly conserved residues from the Y(150)NHNLD motif are described. They have properties similar to those of the wild-type enzyme with respect to their cluster content and characteristics. For all of them, the as-isolated form, which contains an air-stable [2Fe-2S](2+) center, can additionally accommodate an air-sensitive [4Fe-4S](2+) center which is generated by incubation under anaerobic conditions with Fe(2+) and S(2-). Their spectroscopic properties are similar to those of the wild type. However, they are inactive, except the mutant H152A that exhibits a weak activity. We show that the mutants, inactive in producing biotin, are also unable to cleave AdoMet and to produce the deoxyadenosyl radical (AdoCH(2)(*)). In the case of H152A, a value of 5.5 +/- 0.4 is found for the 5'-deoxyadenosine (AdoCH(3)):biotin ratio, much higher than the value of 2.8 +/- 0.3 usually observed with the wild type. This reveals a greater contribution of the abortive process in which the AdoCH(2)(*) radical is quenched by hydrogen atoms from the protein or from some components of the system. Thus, in this case, the coupling between the production of AdoCH(2)(*) and its reaction with the hydrogen at C-6 and C-9 of DTB is less efficient than that in the wild type, probably because of geometry's perturbation within the active site.     

Bifunctional alkylating agents derived from duocarmycin SA: potent antitumor activity with altered sequence selectivity

The series of four dimers derived from head to tail coupling of the two enantiomers of the duocarmycin SA alkylation subunit are described.     

Storage globulins pass through the Golgi apparatus and multivesicular bodies in the absence of dense vesicle formation during early stages of cotyledon development in mung bean

During seed development and maturation, large amounts of storage proteins are synthesized and deposited in protein storage vacuoles (PSVs). Multiple mechanisms have been proposed to be responsible for transporting storage proteins to PSVs in developing seeds. In this study, a specific antibody was raised against the mung bean (Vigna radiata) seed storage protein 8S globulin and its deposition was followed via immunogold electron microscopy in developing mung bean cotyledons. It is demonstrated that non-aggregated 8S globulins are present in multivesicular bodies (MVBs) in early stages of cotyledon development where neither dense vesicles (DVs) nor a PSV were recognizable. However, at later stages of cotyledon development, condensed globulins were visible in both DVs and distinct MVBs with a novel form of partitioning, with the internal vesicles being pushed to one sector of this organelle. These distinct MVBs were no longer sensitive to wortmannin. This study thus indicates a possible role for MVBs in transporting storage proteins to PSVs during the early stage of seed development prior to the involvement of DVs. In addition, wortmannin treatment is shown to induce DVs to form aggregates and to fuse with the plasma membrane.     

Autocrine and paracrine actions of ATP in rat carotid body

Carotid bodies are peripheral chemoreceptors that detect lowering of arterial blood O(2) level. The carotid body comprises clusters of glomus (type I) cells surrounded by glial-like sustentacular (type II) cells. Hypoxia triggers depolarization and cytosolic [Ca(2+)] ([Ca(2+)](i)) elevation in glomus cells, resulting in the release of multiple transmitters, including ATP. While ATP has been shown to be an important excitatory transmitter in the stimulation of carotid sinus nerve, there is considerable evidence that ATP exerts autocrine and paracrine actions in carotid body. ATP acting via P2Y(1) receptors, causes hyperpolarization in glomus cells and inhibits the hypoxia-mediated [Ca(2+)](i) rise. In contrast, adenosine (an ATP metabolite) triggers depolarization and [Ca(2+)](i) rise in glomus cells via A(2A) receptors. We suggest that during prolonged hypoxia, the negative and positive feedback actions of ATP and adenosine may result in an oscillatory Ca(2+) signal in glomus cells. Such mechanisms may allow cyclic release of transmitters from glomus cells during prolonged hypoxia without causing cellular damage from a persistent [Ca(2+)](i) rise. ATP also stimulates intracellular Ca(2+) release in sustentacular cells via P2Y(2) receptors. The autocine and paracrine actions of ATP suggest that ATP has important roles in coordinating chemosensory transmission in the carotid body.