bluuebunny/crossref_metadata_2025 huggingface dataset:
The RCAN carboxyl end mediates calcineurin docking-dependent inhibition via a site that dictates binding to substrates and regulators
Authors: Martínez-Martínez Sara , Genescà Lali , Rodríguez Antonio , Raya Alicia , Salichs Eulàlia , Were Felipe , López-Maderuelo María Dolores , Redondo Juan Miguel , de la Luna Susana
Published: March 2009

Specificity of signaling kinases and phosphatases toward their targets is usually mediated by docking interactions with substrates and regulatory proteins. Here, we characterize the motifs involved in the physical and functional interaction of the phosphatase calcineurin with a group of modulators, the RCAN protein family. Mutation of key residues within the hydrophobic docking-cleft of the calcineurin catalytic domain impairs binding to all human RCAN proteins and to the calcineurin interacting proteins Cabin1 and AKAP79. A valine-rich region within the RCAN carboxyl region is essential for binding to the docking site in calcineurin. Although a peptide containing this sequence compromises NFAT signaling in living cells, it does not inhibit calcineurin catalytic activity directly. Instead, calcineurin catalytic activity is inhibited by a motif at the extreme C-terminal region of RCAN, which acts in
cis
with the docking motif. Our results therefore indicate that the inhibitory action of RCAN on calcineurin-NFAT signaling results not only from the inhibition of phosphatase activity but also from competition between NFAT and RCAN for binding to the same docking site in calcineurin. Thus, competition by substrates and modulators for a common docking site appears to be an essential mechanism in the regulation of Ca
2+
-calcineurin signaling.
Neuronal M3muscarinic acetylcholine receptors are essential for somatotroph proliferation and normal somatic growth
Authors: Gautam Dinesh , Jeon Jongrye , Starost Matthew F. , Han Sung-Jun , Hamdan Fadi F. , Cui Yinghong , Parlow Albert F. , Gavrilova Oksana , Szalayova Ildiko , Mezey Eva , Wess Jürgen
Published: March 2009

The molecular pathways that promote the proliferation and maintenance of pituitary somatotrophs and other cell types of the anterior pituitary gland are not well understood at present. However, such knowledge is likely to lead to the development of novel drugs useful for the treatment of various human growth disorders. Although muscarinic cholinergic pathways have been implicated in regulating somatotroph function, the physiological relevance of this effect and the localization and nature of the receptor subtypes involved in this activity remain unclear. We report the surprising observation that mutant mice that selectively lack the M3muscarinic acetylcholine receptor subtype in the brain (neurons and glial cells; Br-M3-KO mice) showed a dwarf phenotype associated with a pronounced hypoplasia of the anterior pituitary gland and a marked decrease in pituitary and serum growth hormone (GH) and prolactin. Remarkably, treatment of Br-M3-KO mice with CJC-1295, a synthetic GH-releasing hormone (GHRH) analog, rescued the growth deficit displayed by Br-M3-KO mice by restoring normal pituitary size and normal serum GH and IGF-1 levels. These findings, together with results from M3receptor/GHRH colocalization studies and hypothalamic hormone measurements, support a model in which central (hypothalamic) M3receptors are required for the proper function of hypothalamic GHRH neurons. Our data reveal an unexpected and critical role for central M3receptors in regulating longitudinal growth by promoting the proliferation of pituitary somatotroph cells.
Eμ-
TCL1
mice represent a model for immunotherapeutic reversal of chronic lymphocytic leukemia-induced T-cell dysfunction
Authors: Gorgun Gullu , Ramsay Alan G. , Holderried Tobias A. W. , Zahrieh David , Le Dieu Rifca , Liu Fenglong , Quackenbush John , Croce Carlo M. , Gribben John G.
Published: March 2009

Preclinical animal models have largely ignored the immune-suppressive mechanisms that are important in human cancers. The identification and use of such models should allow better predictions of successful human responses to immunotherapy. As a model for changes induced in nonmalignant cells by cancer, we examined T-cell function in the chronic lymphocytic leukemia (CLL) Eμ-
TCL1
transgenic mouse model. With development of leukemia, Eμ-
TCL1
transgenic mice developed functional T-cell defects and alteration of gene and protein expression closely resembling changes seen in CLL human patients. Furthermore, infusion of CLL cells into young Eμ-
TCL1
mice induced defects comparable to those seen in mice with developed leukemia, demonstrating a causal relationship between leukemia and the T-cell defects. Altered pathways involved genes regulating actin remodeling, and T cells exhibited dysfunctional immunological synapse formation and T-cell signaling, which was reversed by the immunomodulatory drug lenalidomide. These results further demonstrate the utility of this animal model of CLL and define a versatile model to investigate both the molecular mechanisms of cancer-induced immune suppression and immunotherapeutic repair strategies.
Adolescent literacy and identity construction among 1.5 generation students
Authors: Yi Youngjoo
Published: March 2009

The emergence and significance of transnational adolescents at school and in society have recently been recognized, and yet, little is known about how their transnational lived experiences affect their literacy learning and identity construction. Thus, the study reported in this paper explored transnational literacy options and practices that two Korean transnational adolescents had experienced and addressed how their online literacy practices served them while negotiating their transnational identities. The findings show that the participants engaged in multiple literacy practices and forged transnational identities through online activities involving “creating and constructing a transnational and transcultural community” and “communicating via instant messaging.” The findings suggest that we should re-conceptualize the teaching and learning of students who share multilingual, transnational lived experiences and that we should re-examine what it means to be good, educated students and global citizens in the 21st century.
Loss of STAT5 causes liver fibrosis and cancer development through increased TGF-β and STAT3 activation
Authors: Hosui Atsushi , Kimura Akiko , Yamaji Daisuke , Zhu Bing-mei , Na Risu , Hennighausen Lothar
Published: March 2009

The molecular mechanisms underlying the development of hepatocellular carcinoma are not fully understood. Liver-specific signal transducer and activator of transcription (STAT) 5A/B–null mice (STAT5-LKO) were treated with carbon tetrachloride (CCl4), and histological analyses revealed liver fibrosis and tumors. Transforming growth factor (TGF)–β levels and STAT3 activity were elevated in liver tissue from STAT5-LKO mice upon CCl4 treatment. To define the molecular link between STAT5 silencing and TGF-β up-regulation, as well as STAT3 activation, we examined STAT5-null mouse embryonic fibroblasts and primary hepatocytes. These cells displayed elevated TGF-β protein levels, whereas messenger RNA levels remained almost unchanged. Protease inhibitor studies revealed that STAT5 deficiency enhanced the stability of mature TGF-β. Immunoprecipitation and immunohistochemistry analyses demonstrated that STAT5, through its N-terminal sequences, could bind to TGF-β and that retroviral-mediated overexpression of STAT5 decreased TGF-β levels. To confirm the in vivo significance of the N-terminal domain of STAT5, we treated mice that expressed STAT5 lacking the N terminus (STAT5-ΔN) with CCl4. STAT5-ΔN mice developed CCl4-induced liver fibrosis but no tumors. In conclusion, loss of STAT5 results in elevated TGF-β levels and enhanced growth hormone–induced STAT3 activity. We propose that a deregulated STAT5–TGF-β–STAT3 network contributes to the development of chronic liver disease.
Ly49H signaling through DAP10 is essential for optimal natural killer cell responses to mouse cytomegalovirus infection
Authors: Orr Mark T. , Sun Joseph C. , Hesslein David G.T. , Arase Hisashi , Phillips Joseph H. , Takai Toshiyuki , Lanier Lewis L.
Published: March 2009

The activating natural killer (NK) cell receptor Ly49H recognizes the mouse cytomegalovirus (MCMV) m157 glycoprotein expressed on the surface of infected cells and is required for protection against MCMV. Although Ly49H has previously been shown to signal via DAP12, we now show that Ly49H must also associate with and signal via DAP10 for optimal function. In the absence of DAP12, DAP10 enables Ly49H-mediated killing of m157-bearing target cells, proliferation in response to MCMV infection, and partial protection against MCMV. DAP10-deficient Ly49H+ NK cells, expressing only Ly49H–DAP12 receptor complexes, are partially impaired in their ability to proliferate during MCMV infection, display diminished ERK1/2 activation, produce less IFN-γ upon Ly49H engagement, and demonstrate reduced control of MCMV infection. Deletion of both DAP10 and DAP12 completely abrogates Ly49H surface expression and control of MCMV infection. Thus, optimal NK cell–mediated immunity to MCMV depends on Ly49H signaling through both DAP10 and DAP12.
The Impact of Ertapenem Use on the Susceptibility of Pseudomonas aeruginosa to Imipenem A Hospital Case Study
Authors: Lima Ana Lucia L. , Oliveira Priscila R. , Paula Adriana P. , Dal-Paz Karine , Rossi Flavia , Zumiotti Arnaldo V.
Published: March 2009

We sought to evaluate the indirect impact of ertapenem use for the treatment of extended-spectrum β-lactamase-producing Enterobacteriaceae infections in our hospital on the susceptibility of Pseudomonas aeruginosa to imipenem. The use of ertapenem was mandated for treatment of extended-spectrum β-lactamase-producing Enterobacteriaceae infections in the absence of nonfermenting gram-negative bacilli for 1 year. The use of imipenem was restricted. Imipenem consumption decreased 64.5%. Ertapenem consumption was 42.57 defined daily doses per 1,000 patient-days. None of the 18 P. aeruginosa isolates recovered after ertapenem introduction were imipenem-resistant, compared with 4 of the 20 P. aeruginosa isolates recovered in the previous year.
Incidence of Microperforation for Surgical Gloves Depends on Duration of Wear
Authors: Partecke Lars Ivo , Goerdt Anna-Maria , Langner Inga , Jaeger Bernd , Assadian Ojan , Heidecke Claus-Dieter , Kramer Axel , Huebner Nils-Olaf
Published: March 2009

Background.

The use of sterile gloves is part of general aseptic procedure, which aims to prevent surgical team members from transmitting infectious agents to patients during procedures performed in an operating room. In addition, surgical gloves also protect team members against patient-transmitted infectious agents. Adequate protection, however, requires that the glove material remain intact. The risk of perforations in surgical gloves is thought to correlate with the duration of wear, yet very few prospective studies have addressed this issue.

Methods.

We prospectively collected 898 consecutive pairs of used surgical gloves over a 9-month period in a single institution. After surgical team members wore the gloves during surgical procedures, the gloves were examined for microperforations using the watertight test described in European Norm 455, part 1. The gloves were analyzed as a pair; if 1 glove had a perforation, the pair was considered to be perforated. In addition, we evaluated the use of a hand cream that contained a suspension of cornstarch and ethanol to determine its potential influence on the rate of microperforation.

Results.

Wearing gloves for 90 minutes or less resulted in microperforations in 46 (15.4%) of 299 pairs of gloves, whereas wearing gloves for 91-150 minutes resulted in perforation of 54 (18.1%) of 299 pairs, and 71 of (23.7%) of 300 pairs were perforated when the duration of wear was longer than 150 minutes (P = .05). Subgroup analysis revealed no significant difference in the rates of microperforation for surgeons (56 [23.0%] of 244 pairs of gloves perforated), first assistants (43 [19.0%] of 226 pairs perforated), and surgical nurses (53 [20.5%] of 259 pairs perforated). Of 171 microperforations, 114 (66.7%) were found on the left hand glove (ie, the glove on subjects' nondominant hand), predominantly on the left index finger (55 [32.3%]). The use of the hand cream had no influence on the rate of microperforation.

Conclusion.

Because of the increase in the rate of microperforation over time, it is recommended that surgeons, first assistants, and surgical nurses directly assisting in the operating field change gloves after 90 minutes of surgery.
Hierarchical control of virginiamycin production in Streptomyces virginiae by three pathway-specific regulators: VmsS, VmsT and VmsR
Authors: Pulsawat Nattika , Kitani Shigeru , Fukushima Eriko , Nihira Takuya
Published: March 2009

Two regulatory genes encoding a Streptomyces antibiotic regulatory protein (vmsS) and a response regulator (vmsT) of a bacterial two-component signal transduction system are present in the left-hand region of the biosynthetic gene cluster of the antibiotic virginiamycin, which is composed of virginiamycin M (VM) and virginiamycin S (VS), in Streptomyces virginiae. Disruption of vmsS abolished both VM and VS biosynthesis, with drastic alteration of the transcriptional profile for virginiamycin biosynthetic genes, whereas disruption of vmsT resulted in only a loss of VM biosynthesis, suggesting that vmsS is a pathway-specific regulator for both VM and VS biosynthesis, and that vmsT is a pathway-specific regulator for VM biosynthesis alone. Gene expression profiles determined by semiquantitative RT-PCR on the virginiamycin biosynthetic gene cluster demonstrated that vmsS controls the biosynthetic genes for VM and VS, and vmsT controls unidentified gene(s) of VM biosynthesis located outside the biosynthetic gene cluster. In addition, transcriptional analysis of a deletion mutant of vmsR located in the clustered regulatory region in the virginiamycin cluster (and which also acts as a SARP-family activator for both VM and VS biosynthesis) indicated that the expression of vmsS and vmsT is under the control of vmsR, and vmsR also contributes to the expression of VM and VS biosynthetic genes, independent of vmsS and vmsT. Therefore, coordinated virginiamycin biosynthesis is controlled by three pathway-specific regulators which hierarchically control the expression of the biosynthetic gene cluster.
Localization and assembly of proteins comprising the outer structures of the Bacillus anthracis spore
Authors: Giorno Rebecca , Mallozzi Michael , Bozue Joel , Moody Krishna-Sulayman , Slack Alex , Qiu Dengli , Wang Rong , Friedlander Arthur , Welkos Susan , Driks Adam
Published: March 2009

Bacterial spores possess a series of concentrically arranged protective structures that contribute to dormancy, survival and, ultimately, germination. One of these structures, the coat, is present in all spores. InBacillus anthracis, however, the spore is surrounded by an additional, poorly understood, morphologically complex structure called the exosporium. Here, we characterize three previously discovered exosporium proteins called ExsFA (also known as BxpB), ExsFB (a highly related paralogue ofexsFA/bxpB) and IunH (similar to an inosine–uridine-preferring nucleoside hydrolase). We show that in the absence of ExsFA/BxpB, the exosporium protein BclA accumulates asymmetrically to the forespore pole closest to the midpoint of the sporangium (i.e. the mother-cell-proximal pole of the forespore), instead of uniformly encircling the exosporium. ExsFA/BxpB may also have a role in coat assembly, as mutant spore surfaces lack ridges seen in wild-type spores and have a bumpy appearance. ExsFA/BxpB also has a modest but readily detected effect on germination. Nonetheless, anexsFA/bxpBmutant strain is fully virulent in both intramuscular and aerosol challenge models in Guinea pigs. We show that the pattern of localization of ExsFA/BxpB–GFP is a ring, consistent with a location for this protein in the basal layer of the exosporium. In contrast, ExsFB–GFP fluorescence is a solid oval, suggesting a distinct subcellular location for ExsFB–GFP. We also used these fusion proteins to monitor changes in the subcellular locations of these proteins during sporulation. Early in sporulation, both fusions were present throughout the mother cell cytoplasm. As sporulation progressed, GFP fluorescence moved from the mother cell cytoplasm to the forespore surface and formed either a ring of fluorescence, in the case of ExsFA/BxpB, or a solid oval of fluorescence, in the case of ExsFB. IunH–GFP also resulted in a solid oval of fluorescence. We suggest the interpretation that at least some ExsFB–GFP and IunH–GFP resides in the region between the coat and the exosporium, called the interspace.
Gene expression patterns and differential input into curli fimbriae regulation of all GGDEF/EAL domain proteins in Escherichia coli
Authors: Sommerfeldt Nicole , Possling Alexandra , Becker Gisela , Pesavento Christina , Tschowri Natalia , Hengge Regine
Published: March 2009

Switching from the motile planktonic bacterial lifestyle to a biofilm existence is stimulated by the signalling molecule bis-(3′-5′)-cyclic-diguanosine monophosphate (cyclic-di-GMP), which is antagonistically controlled by diguanylate cyclases (DGCs; characterized by GGDEF domains) and specific phosphodiesterases (PDEs; mostly featuring EAL domains). Here, we present the expression patterns of all 28 genes that encode GGDEF/EAL domain proteins in Escherichia coli K-12. Twenty-one genes are expressed in Luria–Bertani medium, with 15 being under σ
S control. While a small subset of GGDEF/EAL proteins (YeaJ and YhjH) is dominant and modulates motility in post-exponentially growing cells, a diverse battery of GGDEF/EAL proteins is deployed during entry into stationary phase, especially in cells grown at reduced temperature (28 °C). This suggests that multiple signal input into cyclic-di-GMP control is particularly important in growth-restricted cells in an extra-host environment. Six GGDEF/EAL genes differentially control the expression of adhesive curli fimbriae. Besides the previously described ydaM, yciR, yegE and yhjH genes, these are yhdA (csrD), which stimulates the expression of the DGC YdaM and the major curli regulator CsgD, and yeaP, which contributes to expression of the curli structural operon csgBAC. Finally, we discuss why other GGDEF/EAL domain-encoding genes, despite being expressed, do not influence motility and/or curli formation.
ParB deficiency in Pseudomonas aeruginosa destabilizes the partner protein ParA and affects a variety of physiological parameters
Authors: Bartosik A. A. , Mierzejewska J. , Thomas C. M. , Jagura-Burdzy G.
Published: March 2009

Deletions leading to complete or partial removal of ParB were introduced into thePseudomonas aeruginosachromosome. Fluorescence microscopy of fixed cells showed that ParB mutants lacking the C-terminal domain or HTH motif formed multiple, less intense foci scattered irregularly, in contrast to the one to four ParB foci per cell symmetrically distributed in wild-typeP. aeruginosa. AllparBmutations affected both bacterial growth and swarming and swimming motilities, and increased the production of anucleate cells. Similar effects were observed after inactivation ofparAofP. aeruginosa. As complete loss of ParA destabilized its partner ParB it was unclear deficiency of which protein is responsible for the mutant phenotypes. Analysis of fourparBmutants showed that complete loss of ParB destabilized ParA whereas three mutants that retained the N-terminal 90 aa of ParB did not. As all fourparBmutants demonstrate the same defects it can be concluded that either ParB, or ParA and ParB in combination, plays an important role in nucleoid distribution, growth and motility inP. aeruginosa.
Inactivation of the hmgA gene of Pseudomonas aeruginosa leads to pyomelanin hyperproduction, stress resistance and increased persistence in chronic lung infection
Authors: Rodríguez-Rojas Alexandro , Mena Ana , Martín Soledad , Borrell Nuria , Oliver Antonio , Blázquez Jesús
Published: March 2009

Clinical isolates of Pseudomonas aeruginosa that hyperproduce a dark-brown pigment are quite often found in the lungs of chronically infected patients, suggesting that they may have an adaptive advantage in chronic infections. We have screened a library of random transposon insertions in P. aeruginosa. Transposon insertions resulting in the hyperproduction of a dark-brown pigment were found to be located in the hmgA gene, which putatively encodes the enzyme homogentisate-1,2-dioxygenase. Complementation studies indicate that hmgA disruption is responsible for the hyperproduction of pyomelanin in both laboratory and clinical isolates. A relationship between hmgA disruption and adaptation to chronic infection was explored and our results show that the inactivation of hmgA produces a slight reduction of killing ability in an acute murine model of lung infection. On the other hand, it also confers decreased clearance and increased persistence in chronic lung infections. Whether pyomelanin production is the cause of the increased adaptation to chronicity or just a side effect of hmgA inactivation is a question to be studied in future; however, this adaptation is consistent with the higher resistance to oxidative stress conferred in vitro by the pyomelanin pigment. Our results clearly demonstrate that hmgA inactivation leads to a better adaptation to chronic infection, and strongly suggest that this mechanism may be exploited in naturally occurring P. aeruginosa strains.
Global transcriptional response to vancomycin in Mycobacterium tuberculosis
Authors: Provvedi Roberta , Boldrin Francesca , Falciani Francesco , Palù Giorgio , Manganelli Riccardo
Published: March 2009

In order to gain additional understanding of the physiological mechanisms used by bacteria to maintain surface homeostasis and to identify potential targets for new antibacterial drugs, we analysed the variation of the Mycobacterium tuberculosis transcriptional profile in response to inhibitory and subinhibitory concentrations of vancomycin. Our analysis identified 153 genes differentially regulated after exposing bacteria to a concentration of the drug ten times higher than the MIC, and 141 genes differentially expressed when bacteria were growing in a concentration of the drug eightfold lower than the MIC. Hierarchical clustering analysis indicated that the response to these different conditions is different, although with some overlap. This approach allowed us to identify several genes whose products could be involved in the protection from antibiotic stress targeting the envelope and help to confer the basal level of M. tuberculosis resistance to antibacterial drugs, such as Rv2623 (UspA-like), Rv0116c, PE20-PPE31, PspA and proteins related to toxin–antitoxin systems. Moreover, we also demonstrated that the alternative sigma factor σ
E confers basal resistance to vancomycin, once again underlining its importance in the physiology of the mycobacterial surface stress response.
Lipopolysaccharide biosynthesis-related genes are required for colony pigmentation of Porphyromonas gingivalis
Authors: Sato Keiko , Kido Nobuo , Murakami Yukitaka , Hoover Charles I. , Nakayama Koji , Yoshimura Fuminobu
Published: March 2009

The periodontopathic bacteriumPorphyromonas gingivalisforms pigmented colonies when incubated on blood agar plates as a result of accumulation of μ-oxo haem dimer on the cell surface. Gingipain–adhesin complexes are responsible for production of μ-oxo haem dimer from haemoglobin. Non-pigmented mutants (Tn6-5, Tn7-1, Tn7-3 and Tn10-4) were isolated fromP. gingivalisby Tn4351transposon mutagenesis [Hoover & Yoshimura (1994),FEMS Microbiol Lett124, 43–48]. In this study, we found that the Tn6-5, Tn7-1 and Tn7-3 mutants carried Tn4351DNA in a gene homologous to theugdAgene encoding UDP-glucose 6-dehydrogenase, a gene encoding a putative group 1 family glycosyltransferase and a gene homologous to therfagene encoding ADP heptose-LPS heptosyltransferase, respectively. The Tn10-4 mutant carried Tn4351DNA at the same position as that for Tn7-1. Gingipain activities associated with cells of the Tn7-3 mutant (rfa) were very weak, whereas gingipain activities were detected in the culture supernatants. Immunoblot and mass spectrometry analyses also revealed that gingipains, including their precursor forms, were present in the culture supernatants. A lipopolysaccharide (LPS) fraction of therfadeletion mutant did not show the ladder pattern that was usually seen for the LPS of the wild-typeP. gingivalis. A recombinant chimera gingipain was able to bind to an LPS fraction of the wild-typeP. gingivalisin a dose-dependent manner. These results suggest that therfagene product is associated with biosynthesis of LPS and/or cell-surface polysaccharides that can function as an anchorage for gingipain–adhesin complexes.
Identification of a site-specific tyrosine recombinase that mediates promoter inversions of phase-variable mpl lipoprotein genes in Mycoplasma penetrans
Authors: Horino Atsuko , Kenri Tsuyoshi , Sasaki Yuko , Okamura Noboru , Sasaki Tsuguo
Published: March 2009

Mycoplasma penetrans has the ability to change its surface lipoprotein profiles frequently. The P35 family lipoproteins encoded by the mpl genes are key players in this profile variation. The M. penetrans HF-2 genome has 38 mpl genes that form three gene clusters. Most of these mpl genes have an invertible promoter sequence that is responsible for the ON/OFF switching of individual mpl gene expression. Here, we identified the recombinase that catalyses inversions of the mpl gene promoters. We focused on two open reading frames of the M. penetrans HF-2 genome, namely MYPE2900 and MYPE8180, which show significant homology to the tyrosine site-specific recombinase (Tsr) family proteins. Since genetic tools for M. penetrans are still not developed, we cloned the MYPE2900 and MYPE8180 genes and expressed them in Mycoplasma pneumoniae and Escherichia coli. The promoter regions of the mpl genes [p35 (MYPE6810) or p42 (MYPE6630) genes] were also introduced into M. pneumoniae and E. coli cells expressing MYPE2900 or MYPE8180. Inversion of these promoters occurred in the presence of the MYPE2900 gene but not in the presence of the MYPE8180 gene, indicating that the MYPE2900 gene product is the recombinase that catalyses mpl gene promoter inversions. We used a PCR-based method to detect mpl promoter inversion. This method also enabled us to detect inversions of 10 mpl gene promoters in M. penetrans HF-2 cells. All these promoter inversions occurred at the 12 bp inverted repeat (IR) sequences flanking the promoter sequence. The consensus sequence of these IRs was proposed as TAAYNNNDATTA (Y=C or T; D=A, G or T).
Loss of mannosylphosphate from Candida albicans cell wall proteins results in enhanced resistance to the inhibitory effect of a cationic antimicrobial peptide via reduced peptide binding to the cell surface
Authors: Harris Mark , Mora-Montes Héctor M. , Gow Neil A. R. , Coote Peter J.
Published: March 2009

The outermost layer of theCandida albicanscell wall is enriched with mannosylated glycoproteins. We have used a range of isogenic glycosylation mutants ofC. albicans, which are defective to varying degrees in cell wall protein mannosylation, to investigate the role of the outermost layer of the yeast cell wall in mediating the fungicidal action of the cationic,α-helical antimicrobial peptide dermaseptin S3(1-16) [DsS3(1-16)]. The degree of phosphomannan loss, and concomitant reduction in surface negative charge, from the series of glycosylation mutants correlated with reduced levels of peptide binding to the cells. In turn, the reduced peptide binding correlated with enhanced resistance to DsS3(1-16). To ascertain whether DsS3(1-16) binds to negatively charged phosphate, we studied the effect of exogenous glucosamine 6-phosphate, and glucosamine hydrochloride as a negative control, on the antifungal efficacy of DsS3(1-16). Glucosamine 6-phosphate retarded the efficacy of DsS3(1-16), and this was attributed to the presence of phosphate, because addition of identical concentrations of glucosamine hydrochloride had little detrimental effect on peptide efficacy. Fluorescence microscopy with DsS3(1-16) tagged with fluorescein revealed that the peptide binds to the outer surface of the yeast cell, supporting our previous conclusion that the presence of exterior phosphomannan is a major determinant of the antifungal potency of DsS3(1-16). The binding of the peptide to the cell surface was a transient event that was followed by apparent localization of DsS3(1-16) in the vacuole or dissemination throughout the entire cytosol. The presence of glucosamine 6-phosphate clearly reduced the proportion of cells in the population that showed complete cytosolic staining, implying that the binding and entry of the peptide into the cytosol is significantly reduced due to the exogenous phosphate sequestering the peptide and reducing the amount of peptide able to bind to the surface phosphomannan. In conclusion, we present evidence that an antimicrobial peptide, similar to those employed by cells of the human immune system, has evolved to recognize molecular patterns on the surface of pathogens in order to maximize efficacy.
Multiple H1‐antihistamine‐induced urticaria
Authors: INOMATA Naoko , TATEWAKI Satoko , IKEZAWA Zenro
Published: March 2009

H1‐antihistamines are widely used in the treatment of various allergic diseases. Particularly, a cornerstone of the management of chronic idiopathic urticaria is treatment with H1‐antihistamines. However, a few cases of H1‐antihistamine‐induced urticaria have been reported. A 34‐year‐old woman presented with a 4‐month history of recurrent urticaria, which was prominently exacerbated by the administration of H1‐antihistamines. The patient consented to a provocation test of fexofenadine among drugs including cetirizine and hydroxyzine, which were suspected of inducing severe symptoms in episodes. One hour after challenge with 12 mg fexofenadine (one‐fifth of the therapeutic dose), a urticarial reaction rapidly developed on nearly the entire body with remarkably increased levels of plasma histamine (190 nmol/L) and plasma leukotriene B4 (150 pg/mL). In challenge tests with other antihistamines, generalized urticaria occurred 5 and 1 h after intake of 10 mg loratadine and 10 mg bepotastine, respectively, whereas challenges with chlorpheniramine, mequitazine and azelastine were all negative. Skin prick tests with H1‐antihistamines used in the challenges were all negative, indicating that the urticarial reactions after challenges with the causative drugs might not be immunoglobulin E‐mediated. Among the causative drugs in our case, cetirizine and hydroxyzine are the piperazine derivatives, whereas fexofenadine, bepotastine, ebastine and loratadine are the piperidine derivatives. The chemical structures of both derivatives are very similar. Therefore, in this case, H1‐antihistamine‐induced urticaria may have been due to cross‐reactivity between metabolites of these drugs, but not to drugs before metabolization. Hypersensitivity to H1‐antihistamines should be considered when urticarial lesions worsen after H1‐antihistamine treatment.
Ulcerative pigmented squamous cell carcinoma in a 101‐year‐old Japanese woman
Authors: YOSHIDA Yuichi , YAMASAKI Ayako , SHIOMI Tatsushi , SUYAMA Yoshiko , NAKAYAMA Bin , YAMAMOTO Osamu
Published: March 2009

Pigmented squamous cell carcinoma is a rare variant of squamous cell carcinoma. We report a case of pigmented squamous cell carcinoma with dermoscopic examination probably arisen from actinic keratosis in a 101‐year‐old woman who was surgically treated under general anesthesia. In addition, we discuss indications of general anesthesia in elderly patients with skin cancer, differential diagnosis and dermoscopic features of pigmented squamous cell carcinoma.
Isolation of stem/progenitor cells from normal lung tissue of adult humans
Authors: Tesei A.  , Zoli W.  , Arienti C.  , Storci G.  , Granato A. M.  , Pasquinelli G.  , Valente S.  , Orrico C.  , Rosetti M.  , Vannini I.  , Dubini A.  , Dell’Amore D.  , Amadori D.  , Bonafè M. 
Published: March 2009

Objectives:  This study aimed to isolate and characterize stem/progenitor cells, starting from normal airway epithelia, obtained from human adults.

Materials and methods:  Cultures of multicellular spheroids were obtained from human lung tissue specimens after mechanical and enzymatic digestion. Tissue‐specific markers were detected on their cells by immunohistochemical and immunofluorescent techniques. Ultrastructural morphology of the spheroids (termed as bronchospheres) was evaluated by electron microscopy, gene expression analysis was performed by reverse transcription–polymerase chain reaction, and gene down‐regulation was analysed by an RNA interference technique.

Results:  Bronchospheres were found to be composed of cells with high expression of stem cell regulatory genes, which was not or was only weakly detectable in original tissues. Morphological analysis showed that bronchospheres were composed of mixed phenotype cells with type II alveolar and Clara cell features, highlighting their airway resident cell origin. In addition to displaying specific pulmonary and epithelial commitment, bronchospheres showed mesenchymal features. Silencing of the Slug gene, known to play a pivotal role in epithelial–mesenchymal transition processes and which was highly expressed in bronchospheres but not in original tissue, led bronchospheres to gain a differentiated bronchial/alveolar phenotype and to lose the stemness gene expression pattern.

Conclusions:  Ours is the first study to describe ex vivo expansion of stem/progenitor cells resident in human lung epithelia, and our results suggest that the epithelial–mesenchymal transition process, still active in a subset of airway cells, may regulate transit of stem/progenitor cells towards epithelial differentiation.
Detecting ecological responses to flow variation using Bayesian hierarchical models
Authors: WEBB J. ANGUS , STEWARDSON MICHAEL J. , KOSTER WAYNE M.
Published: March 2009

1. Inferring effects of environmental flows is difficult with standard statistical approaches because flow‐delivery programs are characterised by weak experimental design, and monitoring programs often have insufficient replication to detect ecologically significant effects. Bayesian hierarchical approaches may be more suited to the task, as they are more flexible and allow data from multiple non‐replicate sampling units (e.g. rivers) to be combined, increasing inferential strength.

2. We assessed the utility of Bayesian hierarchical models for detecting ecological effects of flow variation by conducting both hierarchical and non‐hierarchical analyses on two environmental endpoints. We analysed effects of discharge on salinity in the Wimmera and Glenelg rivers (Victoria, Australia) using a linear regression with autocorrelation terms, and on Australian smelt in the Thomson River (Victoria, Australia) using a multi‐level covariate model. These analyses test some of the hypotheses upon which environmental flow recommendations have been made for these rivers.

3. Discharge was correlated with reduced salinity at six of 10 sites, but with increased salinity at two others. The results were very similar for hierarchical and non‐hierarchical models. For Australian smelt, the hierarchical model found some evidence that excess summer discharge reduces abundance in all river reaches, but the non‐hierarchical model was able to detect this response in only one reach.

4. The results highlight the power and flexibility of Bayesian analysis. Neither of the models fitted would have been amenable to more widely used statistical approaches, and it is unlikely that we would have detected responses to flow variation in these data had we been using such techniques. Hierarchical models can greatly improve inferential strength in the data‐poor situations that are common in ecological monitoring, and will be able to be used to assess the effectiveness of environmental flow programs and maximise the benefits of large‐scale environmental flow monitoring programs.
Nesting ecology in the freshwater turtlePodocnemis unifilis: spatiotemporal patterns and inferred explanations
Authors: Escalona Tibisay , Valenzuela Nicole , Adams Dean C.
Published: March 2009

Habitat selection has profound ecological and evolutionary consequences. For example, there may be strong selection for nest‐site choice such that oviparous females, lacking parental care, may adaptively manipulate their offspring survival or phenotype. Alternatively, nesting decisions may be a passive by‐product of other processes leading to similar nonrandom nesting patterns.

Here we examined the nesting ecology of the turtlePodocnemis unifilisat multiple spatiotemporal scales to determine whether randomness, adaptive nest‐site selection or social facilitation best explained the observed population‐level patterns. We addressed these alternative strategies by exploring how environmental and geographic variation in nest sites influences embryonic survival within and across four nesting beaches in three years.

We found nonrandom spatial and environmental patterns of nesting within beaches and years, consistent with both the adaptive nest‐site selection and social facilitation hypotheses. However, nesting patterns were unpredictable among beaches and among years. Furthermore, environmental conditions at nest sites and offspring survival were not associated, and nests from the most gregarious nesting night clustered more tightly geographically and suffered lower predation than nests laid on other nights.

Together, our findings provide more extensive support for social facilitation as compared to the adaptive nest‐site selection hypothesis. Our results suggest that selection for female nest‐site choice in reptiles may be acting more strongly via offspring survival through nest clustering derived from conspecific cueing and less strongly via environmental cueing than previously anticipated.

Our findings underscore the importance of examining multiple sites during multiple seasons. This approach permits testing critical predictions about the consistency of population‐level patterns across space and time that enable the distinction between models.

Our results support a shift with regards to the trait that is usually considered the target of selection for female nest‐site choice in a way that exemplifies the classic dichotomy between selectionfor(survival of nests and perhaps of females) and selectionof(offspring phenotype, such as sex).
A slow life in hell or a fast life in heaven: demographic analyses of contrasting roe deer populations
Authors: Nilsen Erlend B. , Gaillard Jean‐Michel , Andersen Reidar , Odden John , Delorme Daniel , Van Laere Guy , Linnell John D. C.
Published: March 2009

Environmental conditions shape population growth through their impact on demographic parameters. While knowledge has accumulated concerning the effects of population density and climatic conditions, a topical question now concerns how predation and harvest influence demographic parameters and population growth (λ).

We performed a comparative demographic analysis based on projection matrix models for female roe deer. Population‐specific matrices were parameterized based on longitudinal data from five intensively monitored populations in Norway and France, spanning a large variability in environmental characteristics such as densities of large predators, hunter harvest and seasonality.

As expected for a large iteroparous vertebrate, temporal variation was invariably higher in recruitment than in adult survival, and the elasticity of adult survival was consistently higher than that of recruitment. However, the relative difference in elasticity of λ to recruitment and adult survival varied strongly across populations, and was closely correlated with adult survival.

Different traits accounted for most of the variance in λ in different ecological settings. Adult survival generally contributed more in populations with low mean adult survival and low mean growth rate during the study period. Hunters and predators (Eurasian lynx and red foxes) occurred in two of our study populations and contributed substantially to the variance in λ, accounting for a total of 35% and 70% in the two populations respectively.

Across populations, we did not find any evidence that roe deer increased their reproductive output when faced with harsh conditions, resulting in some populations having negative growth rates.

Generation time, a measure of the speed of the life‐history cycle, increased from less than 4 years in the most productive population (‘roe deer heaven’) to more than 6 years in declining populations facing predation from lynx, red fox and hunters (‘roe deer hell’), and was tightly and inversely correlated with λ. Such a deceleration of the life cycle in declining populations might be a general feature in large herbivores.

Our results shows that the plethora of environmental conditions faced by populations of large herbivores also induce high intraspecific variation in their ranking along the ‘fast–slow’ continuum of life‐history tactics.
Combined oesophageal impedance‐pH monitoring in preterm newborn: comparison of two options for layout analysis
Authors: Corvaglia L. , Mariani E. , Aceti A. , Capretti M.g. , Ancora G. , Faldella G.
Published: March 2009

Abstract  Gastro‐oesophageal reflux (GOR) is common in preterm infants. Combined multichannel intraluminal impedance and pH monitoring (pH‐MII) is emerging as an useful tool to study both acid and non‐acid GOR in this population. We aimed to highlight main advantages and limits of pH‐MII in preterm infants and to test whether the inclusion of GOR episodes detected only by pH monitoring details better the features of GOR. Fifty‐two symptomatic preterm infants underwent a 24‐hour, continuous and simultaneous measurement of pH‐MII. Each layout was analyzed using two different options: option 1 included GOR episodes detected by MII and then classified as acid or non‐acid according to the associated pH change; option 2 included GOR episodes detected by MII and also GOR episodes detected only by pH sensor. By adopting option 1, a total number of 2834 GOR episodes was detected by MII: 2162 of them were characterized as non‐acid and 672 were characterized as acid. The median (range) number of acid MII‐GOR episodes was 10 (1–52); the median (range) number of non‐acid MII‐GOR episodes was 36.5 (2–119). Median (range) acid MII‐GOR‐bolus exposure index was 0.28% (0.02–2.73%); median (range) non‐acid MII‐GOR‐bolus exposure index was 1.03% (0.06–38.15%). By adopting option 2, an average of 53.2 acid GOR episodes and an average of 11% oesophageal exposure to acid GOR more than by option 1 was detected. An accurate and detailed description of GOR in preterm infants can be obtained only by including in the analysis all acid GOR episodes detected by pH sensor.
ATP‐dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice
Authors: Gomes P. , Chevalier J. , Boesmans W. , Roosen L. , Van Den Abbeel V. , Neunlist M. , Tack J. , Vanden Berghe P.
Published: March 2009

Abstract  The importance of dynamic interactions between glia and neurons is increasingly recognized, both in the central and enteric nervous system. However, apart from their protective role, little is known about enteric neuro–glia interaction. The aim was to investigate neuro–glia intercellular communication in a mouse culture model using optical techniques. Complete embryonic (E13) guts were enzymatically dissociated, seeded on coverslips and studied with immunohistochemistry and Ca2+‐imaging. Putative progenitor‐like cells (expressing both PGP9.5 and S‐100) differentiated over approximately 5 days into glia or neurons expressing typical cell‐specific markers. The glia–neuron ratio could be manipulated by specific supplements (N2, G5). Neurons and glia were functionally identified both by their Ca2+‐response to either depolarization (high K+) or lysophosphatidic acid and by the expression of typical markers. Neurons responded to ACh, DMPP, 5‐HT, ATP and electrical stimulation, while glia responded to ATP and ADPβs. Inhibition of glial responses by MRS2179 suggests involvement of P2Y1 receptors. Neuronal stimulation also caused delayed glial responses, which were reduced by suramin and by exogenous apyrases that catalyse nucleotide breakdown. Conversely, glial responses were enhanced by ARL‐67156, an ecto‐ATPase inhibitor. In this mouse enteric co‐culture, functional glia and neurons can be easily monitored using optical techniques. Glial cells can be activated directly by ATP or ADPβs. Activation of neuronal cells (DMPP, K+) causes secondary responses in glial cells, which can be modulated by tuning ATP and ADP breakdown. This strongly supports the involvement of paracrine purinergic communication between enteric neurons and glia.
Persistent measles virus infection of mouse neural cells lacking known human entry receptors
Authors: Abdullah H. , Earle J. A. P. , Gardiner T. A. , Tangy F. , Cosby S. L.
Published: March 2009

Aims: Infection of the mouse central nervous system with wild type (WT) and vaccine strains of measles virus (MV) results in lack of clinical signs and limited antigen detection. It is considered that cell entry receptors for these viruses are not present on murine neural cells and infection is restricted at cell entry. Methods: To examine this hypothesis, virus antigen and caspase 3 expression (for apoptosis) was compared in primary mixed, neural cell cultures infected in vitro or prepared from mice infected intracerebrally with WT, vaccine or rodent neuroadapted viruses. Viral RNA levels were examined in mouse brain by nested and real‐time reverse transcriptase polymerase chain reaction. Results: WT and vaccine strains were demonstrated for the first time to infect murine oligodendrocytes in addition to neurones despite a lack of the known MV cell receptors. Unexpectedly, the percentage of cells positive for viral antigen was higher for WT MV than neuroadapted virus in both in vitro and ex vivo cultures. In the latter the percentage of positive cells increased with time after mouse infection. Viral RNA (total and mRNA) was detected in brain for up to 20 days, while cultures were negative for caspase 3 in WT and vaccine virus infections. Conclusions: WT and vaccine MV strains can use an endogenous cell entry receptor(s) or alternative virus uptake mechanism in murine neural cells. However, viral replication occurs at a low level and is associated with limited apoptosis. WT MV mouse infection may provide a model for the initial stages of persistent MV human central nervous system infections.