bluuebunny/crossref_metadata_2025 huggingface dataset:
Candida albicans biofilm formation in a new in vivo rat model
Authors: Řičicová Markéta , Kucharíková Soňa , Tournu Hélène , Hendrix Jelle , Bujdáková Helena , Van Eldere Johan , Lagrou Katrien , Van Dijck Patrick
Published: December 2009

Device-associated microbial growth, including Candida biofilms, represents more than half of all human microbial infections and, despite a relatively small risk of implant-associated diseases, this type of infection usually leads to high morbidity, increased health-care costs and prolonged antimicrobial therapy. Animal models are needed to elucidate the complex host–pathogen interactions that occur during the development of attached and structured biofilm populations. We describe here a new in vivo model to study Candida biofilm, based on the avascular implantation of small catheters in rats. Polyurethane biomaterials challenged with Candida cells were placed underneath the skin of immunosuppressed animals following only minor surgery. The model allowed the study of up to ten biofilms at once, and the recovery of mature biofilms from 2 days after implantation. The adhering inoculum was adjusted to the standard threshold of positive diagnosis of fungal infection in materials recovered from patients. Wild-type biofilms were mainly formed of hyphal cells, and they were unevenly distributed across the catheter length as observed in infected materials in clinical cases. The hyphal multilayered structure of the biofilms of wild-type strains was observed by confocal microscopy and compared to the monolayer of yeast or hyphal cells of two well-known biofilm-deficient strains, efg1Δ/efg1Δ cph1Δ/cph1Δ and bcr1Δ/bcr1Δ, respectively. The subcutaneous Candida biofilm model relies on the use of implanted catheters with accessible, fast and minor surgery to the animals. This model can be used to characterize the ability of antimicrobial agents to eliminate biofilms, and to evaluate the prophylactic effect of antifungal drugs and biomaterial coatings.
Carotenoids of Gemmatimonas aurantiaca (Gemmatimonadetes): identification of a novel carotenoid, deoxyoscillol 2-rhamnoside, and proposed biosynthetic pathway of oscillol 2,2′-dirhamnoside
Authors: Takaichi Shinichi , Maoka Takashi , Takasaki Kazuto , Hanada Satoshi
Published: December 2009

Gemmatimonas aurantiaca strain T-27T is an orange-coloured, Gram-negative, facultatively aerobic, polyphosphate-accumulating bacterium belonging to a recently proposed phylum, Gemmatimonadetes. We purified its pigments and identified them as carotenoids and their glycoside derivatives using spectral data. The major carotenoid was (2S,2′S)-oscillol 2,2′-di-(α-l-rhamnoside), and the minor carotenoids were (2S)-deoxyoscillol 2-(α-l-rhamnoside) and didemethylspirilloxanthin. Deoxyoscillol2-rhamnoside is a novel carotenoid. Oscillol 2,2′-diglycosides have hitherto only been reported in a limited number of cyanobacteria, and this is believed to be the first finding of such carotenoids in another bacterial phylum. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence, we propose a biosynthetic pathway for the carotenoids and the corresponding genes and enzymes. We propose the involvement of geranylgeranyl pyrophosphate synthase (CrtE), phytoene synthase (CrtB) and phytoene desaturase (CrtI) for lycopene synthesis; and of carotenoid1,2-hydratase (CruF) and carotenoid 2-O-rhamnosyltransferase (CruG) for oscillol 2,2′-dirhamnoside synthesis. Further, isopentenyl pyrophosphate could be synthesized by a non-mevalonate pathway (DXP pathway).
The periplasmic nitrate reductase in Shewanella: the resolution, distribution and functional implications of two NAP isoforms, NapEDABC and NapDAGHB
Authors: Simpson Philippa J. L. , Richardson David J. , Codd Rachel
Published: December 2009

In the bacterial periplasm, the reduction of nitrate to nitrite is catalysed by a periplasmic nitrate reductase (NAP) system, which is a species-dependent assembly of protein subunits encoded by thenapoperon. The reduction of nitrate catalysed by NAP takes place in the 90 kDa NapA subunit, which contains a Mo-bis-molybdopterin guanine dinucleotide cofactor and one [4Fe−4S] iron–sulfur cluster. A review of thenapoperons in the genomes of 19 strains ofShewanellashows that most genomes contain twonapoperons. This is an unusual feature of this genus. The two NAP isoforms each comprise three isoform-specific subunits – NapA, a di-haem cytochrome NapB, and a maturation chaperone NapD – but have different membrane-intrinsic subunits, and have been named NAP-α(NapEDABC) and NAP-β(NapDAGHB). SixteenShewanellagenomes encode both NAP-αand NAP-β. The genome of the vigorous denitrifierShewanella denitrificansOS217 encodes only NAP-αand the genome of the respiratory nitrate ammonifierShewanella oneidensisMR-1 encodes only NAP-β. This raises the possibility that NAP-αand NAP-βare associated with physiologically distinct processes in the environmentally adaptable genusShewanella.
Phenotypic and genomic characterization of pneumococcus-like streptococci isolated from HIV-seropositive patients
Authors: Leegaard Truls M. , Bootsma Hester J. , Caugant Dominique A. , Eleveld Marc J. , Mannsåker Turid , Frøholm Leif Oddvar , Gaustad Peter , Høiby E. Arne , Hermans Peter W. M.
Published: December 2009

Accurate differentiation between pneumococci and other viridans streptococci is essential given their differences in clinical significance. However, classical phenotypic tests are often inconclusive, and many examples of atypical reactions have been reported. In this study, we applied various phenotypic and genotypic methods to discriminate between a collection of 12 streptococci isolated from the upper respiratory tract of HIV-seropositive individuals in 1998 and 1999. Conventional phenotypic characterization initially classified these streptococci as Streptococcus pneumoniae, as they were all sensitive to optochin and were all bile soluble. However, they did not agglutinate with anti-pneumococcal capsular antibodies and were also far more resistant to antimicrobial agents than typeable pneumococci isolated in the same period. Genotypic characterization of these isolates and control isolates by both multilocus sequence analysis (MLSA) and comparative genomic hybridization (CGH) showed that only a single isolate was genetically considered to be a true S. pneumoniae isolate, and that the remaining 11 non-typable isolates were indeed distinct from true pneumococci. Of these, 10 most closely resembled a subgroup of Streptococcus mitis isolates genetically, while one strain was identified as a Streptococcus pseudopneumoniae isolate. CGH also showed that a considerable part of the proposed pneumococcal core genome, including many of the known pneumococcal virulence factors, was conserved in the non-typable isolates. Sequencing of part of the 16S rRNA gene and investigation for the presence of ply by PCR corroborated these results. In conclusion, our findings confirm the close relationship between streptococci of the Mitis group, and show that both MLSA and CGH enable pneumococci to be distinguished from other Mitis group streptococci.
FdTonB is involved in the photoregulation of cellular morphology during complementary chromatic adaptation in Fremyella diplosiphon
Authors: Pattanaik Bagmi , Montgomery Beronda L.
Published: December 2009

We have characterized a Fremyella diplosiphon TonB protein (FdTonB) and investigated its function during complementary chromatic adaptation. Sequence similarity analysis of FdTonB (571 aa) led to identification of several conserved domains characteristic of TonB proteins, including an N-terminal transmembrane domain, a central proline-rich spacer and a C-terminal TonB-related domain (TBRD). We identified a novel glycine-rich domain containing (Gly-X)
n
repeats. To assess FdTonB function, we constructed a ΔtonB mutant through homologous recombination based upon truncation of the central proline-rich spacer, glycine-rich domain and TBRD. Our ΔtonB mutant exhibited an aberrant cellular morphology under green light, with expanded cell width compared to the parental wild-type (WT) strain. The cellular morphology of the ΔtonB mutant recovered upon WT tonB expression. Interestingly, tonB expression was found to be independent of RcaE. As ΔtonB and WT strains respond in the same way when grown under iron-replete versus iron-limited conditions, our results suggest that FdTonB is not involved in the classic TonB function of mediating cellular adaptation to iron limitation, but exhibits a novel function related to the photoregulation of cellular morphology in F. diplosiphon.
Biased usage of BV gene families of T‐cell receptors of WT1 (Wilms’ tumor gene)‐specific CD8+ T cells in patients with myeloid malignancies
Authors: Tanaka‐Harada Yukie , Kawakami Manabu , Oka Yoshihiro , Tsuboi Akihiro , Katagiri Takamasa , Olga Elisseeva , Nishida Sumiyuki , Shirakata Toshiaki , Hosen Naoki , Fujiki Fumihiro , Murao Ayako , Nakajima Hiroko , Oji Yusuke , Kanda Yoshinobu , Kawase Ichiro , Sugiyama Haruo
Published: December 2009

(Cancer Sci 2010; 101: 594–600)

WT1 (Wilms’ tumor gene 1) protein is a potent pan‐tumor‐associated antigen (TAA) and WT1‐specific cytotoxic T lymphocytes (WT1 tetramer+ CD8+ T cells) are spontaneously induced in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). We conducted a single‐cell level comparative analysis of T‐cell receptor β‐chain variable region (TCR‐BV) gene families of a total of 1242 spontaneously induced WT1 tetramer+ CD8+ T cells in HLA‐A*2402+ patients with AML or MDS and those in healthy donors (HDs). This is the first report of direct usage analysis of TCR‐BV gene families of individual TAA‐specific CD8+ T cells at single‐cell level. Usage analysis using single‐cell RT‐PCR of TCR‐BV gene families of individual FACS‐sorted WT1 tetramer+ CD8+ T cells showed for the first time (i) that BVs 5, 6, 20, and 27 were commonly biased in both HDs and patients; (ii) that BV4 was commonly biased in HDs and MDS patients; (iii) that BV19 was commonly biased in the patients; and (iv) that BVs 7 and 28, BVs 9 and 15, and BVs 12 and 29 were specifically biased in HDs, AML, and MDS patients, respectively. However, statistical analysis of similarity among HD, AML, and MDS of individual usage frequencies of 24 kinds of TCR‐BV gene families indicated that the usage frequencies of TCR‐BV gene families in AML and MDS patients reflect those in HDs. These findings represent a novel insight for a better understanding of WT1‐specific immune response.
Augmentation of 3‐methylcholanthrene‐induced bioactivation in the human hepatoma cell line HepG2 by the calcium channel blocker nicardipine
Authors: Hosaka Takuomi , Sekimoto Masashi , Nemoto Kiyomitsu , Degawa Masakuni
Published: December 2009

(Cancer Sci2010; 101: 652–657)

The abilities of the dihydropyridine calcium channel blocker nicardipine (Nic) to induce cytochrome P450 1 family enzymes (CYP1s) and to enhance the 3‐methylcholanthrene (MC)‐mediated induction of CYP1s and formation of MC‐DNA adduct were examined in the human hepatoma cell line HepG2. The results from real time RT‐PCR analysis demonstrated that Nic could induce CYP1 mRNAs and enhance the MC‐mediated induction of the CYP1 mRNAs. The luciferase‐reporter gene assay using the HepG2‐A10 cell line, which has been previously established for the screening of aryl hydrocarbon receptor (AhR) activators, also indicated the augmentation of MC‐mediated activation of AhR (induction of luciferase) by Nic, although Nic showed limited capacity for the activation of AhR. Furthermore, the results from the Western blot analysis of CYP1s, the enzyme activity assay, and the assay for MC‐DNA adduct formation indicated that Nic could enhance the MC‐mediated induction of CYP1s, especially CYP1A1. Furthermore, the intracellular accumulation level of [3H]MC after treatment of HepG2 cells with [3H]MC significantly increased in the presence of Nic. The present findings demonstrate that Nic can enhance the MC‐mediated induction of CYP1s, especially CYP1A1, and the formation of MC‐DNA adduct in HepG2 cells. Furthermore, the augmentation of the MC‐mediated bioactivation by Nic is demonstrated to occur mainly through an increase in intracellular accumulation of MC.
Altered expression of glycan genes in cancers induced by epigenetic silencing and tumor hypoxia: Clues in the ongoing search for new tumor markers
Authors: Kannagi Reiji , Sakuma Keiichiro , Miyazaki Keiko , Lim Khe‐Ti , Yusa Akiko , Yin Jun , Izawa Mineko
Published: December 2009

(Cancer Sci 2010; 101: 586–593)

The glycan molecules that preferentially appear in cancers are clinically utilized as serum tumor markers. The exact reason, however, why glycans are useful as tumor markers remain elusive. Here, we will summarize lessons learned from well‐established cancer‐associated glycans, and propose strategies to develop new cancer markers. Our recent results on cancer‐associated glycans, sialyl Lewis A and sialyl Lewis X, indicated that the repressed transcription of some glycan genes by epigenetic silencing during early carcinogenesis, and the transcriptional induction of some other glycan genes by tumor hypoxia accompanying cancer progression at locally advanced stages, are two major factors determining cancer‐associated glycan expression. Multiple genes are involved in glycan synthesis, and epigenetic silencing of a part of such genes leads to accumulation of glycans having truncated incomplete structures, which are readily detected by specific antibodies. Glycans are very unique and advantageous as marker molecules because they are capable of reflecting epigenetic silencing in their structures. Transcriptional induction of some glycan genes by tumor hypoxia at the later stages produces further glycan modifications, such as an unusual increase of the N‐glycolyl sialic acid residues in the glycan molecules. The entire process of malignant transformation thus creates abnormal glycans, whose structures reveal the effects of both epigenetic silencing and tumor hypoxia. The second advantage of a glycan marker over a proteinous marker is that they can reflect the plurality of genetic anomalies in a singular molecule, as it is synthesized by the cooperative action of multiple genes. Glycans are sometimes covalently bound to well‐known cancer‐associated proteins, such as CD44v, and this eventually contributes to a high cancer specificity and functional relevancy in cancer progression.
Age dependence of radiation‐induced renal cell carcinomas in an Eker rat model
Authors: Kokubo Toshiaki , Kakinuma Shizuko , Kobayashi Toshiyuki , Watanabe Fumiko , Iritani Riichirou , Tateno Kaori , Nishimura Mayumi , Nishikawa Tetsu , Hino Okio , Shimada Yoshiya
Published: December 2009

(Cancer Sci 2010; 101: 616–623)

Exposure to carcinogens early in life may contribute to cancer development later in life. The amount of radiation exposure children experience during medical procedures has been increasing, so it is important to evaluate the radiation risk of cancer in developing organs. Toward this goal, we assessed the risk of developing renal cell carcinoma using Eker rats as a kidney tumor model. F1 hybrids of male Eker (Tsc2 mutant) and female F344 rats were irradiated with 0.5 or 2 Gy gamma radiation on gestation days 15 and 19, and on postnatal days 5, 20, and 49. At 27 weeks of age, kidneys were examined for proliferative lesions. Preneoplastic lesions such as phenotypically altered tubules increased after postnatal irradiation as a function of age‐at‐irradiation, and hyperplasia were greatly increased after perinatal and postnatal irradiation. In contrast, development of adenoma and adenocarcinoma were evident in animals irradiated at perinatal ages, being maximal at gestational day 19. The frequency of LOH at the Tsc2 locus was unexpectedly low – 0% (0 of 4) for the unirradiated control, and 17% (6 of 35) for the irradiated group. Irrespective of LOH, the mTOR (mammalian target of rapamycin) pathway, which is negatively regulated by the Tsc1/2 complex, was activated in both benign and malignant lesions, as evidenced by phosphorylation of S6 ribosomal protein and 4E‐BP1. This suggests that the wild‐type Tsc2 allele may be functionally inactivated. In conclusion, actively growing kidneys in perinatal‐aged (F344 × Eker) F1 rats (Tsc2+/−) are at risk for radiation‐induced malignant transformation of the renal epithelium associated with mTOR activation.
High expression of BUBR1 is one of the factors for inducing DNA aneuploidy and progression in gastric cancer
Authors: Ando Koji , Kakeji Yosihiro , Kitao Hiroyuki , Iimori Makoto , Zhao Yan , Yoshida Rintaro , Oki Eiji , Yoshinaga Keiji , Matumoto Takuya , Morita Masaru , Sakaguchi Yoshihisa , Maehara Yoshihiko
Published: December 2009

(Cancer Sci 2010; 101: 639–645)

Gastric cancers show high frequency of DNA aneuploidy, a phenotype of chromosomal instability. It is suggested that the abnormal spindle assembly checkpoint is involved in DNA aneuploidy, but the underlying mechanism is still unclear. We studied the mechanism by assessing the expression of BUBR1 in gastric cancer. The DNA ploidy patterns of 116 gastric cancer samples obtained from the Department of Surgery and Science at Kyushu University Hospital were analyzed. Of those, DNA aneuploidy was seen in 70 (60.3%) cases of gastric cancer. The expression of BUBR1 was studied by immunohistochemistry in 181 gastric cancer samples and by real‐time RT‐PCR in several gastric cancer cell lines. Ninety‐one (50.3%) cases had high expression of BUBR1 and those cases correlated significantly with DNA aneuploidy (P < 0.05). Also high expression of BUBR1 cases had significant correlation with deep invasion, lymph node metastasis, liver metastasis, and poor prognosis. In gastric cancer cell lines, high expression of BUBR1 had a significant relationship with DNA aneuploidy (P < 0.05). Then, gastric cancer cell lines MKN‐28 and SNU‐1 were transfected with full‐length BUBR1 to observe the significance of the change in BUBR1 expression. Enforced expression of BUBR1 resulted in changes to the ploidy pattern and high Ki‐67 expression. Collectively, our clinical and in vitro data indicate that high expression of BUBR1 may be one of causative factors for the induction of DNA aneuploidy and progression of gastric cancer.
Advances in the understanding and management of angioimmunoblastic T‐cell lymphoma
Authors: De Leval Laurence , Gisselbrecht Christian , Gaulard Philippe
Published: December 2009

Angioimmunoblastic T‐cell lymphoma (AITL) is a distinct peripheral T‐cell lymphoma (PTCL) entity with peculiar clinical and pathological features. The recent identification of follicular helper T (TFH) cell as the cell of origin of this neoplasm represents a major step in our understanding of the pathobiological characteristics of the disease and should, in the future, clarify the diagnostic criteria for AITL and help to delineate its spectrum, especially from PTCL, not otherwise specified (PTCL, NOS). Deciphering the pathogenesis of the disease is needed to identify targets for new therapies that are expected to improve the poor outcome of AITL patients, when treated with conventional chemotherapy regimens. In this respect, efforts will be needed to evaluate promising innovative therapies in prospective clinical trials.
Role of 5‐HT2A, 5‐HT4and 5‐HT7receptors in the antigen‐induced airway hyperresponsiveness in guinea‐pigs
Authors: Segura P. , Vargas M. H. , Córdoba‐Rodríguez G. , Chávez J. , Arreola J. L. , Campos‐Bedolla P. , Ruiz V. , García‐Hernández L. M. , Méndez C. , Montaño L. M.
Published: December 2009

BackgroundA possible role of 5‐hydroxytryptamine (5‐HT) in the origin of antigen‐induced airway hyperresponsiveness (AI‐AHR) has been scarcely investigated.

ObjectiveTo explore the participation of different 5‐HT receptors in the development of AI‐AHR in guinea‐pigs.

MethodsLung resistance was measured in anaesthetized guinea‐pigs sensitized to ovalbumin (OVA). Dose–response curves to intravenous (i.v.) acetylcholine (ACh) were performed before and 1 h after antigenic challenge and expressed as the 200% provocative dose (PD200). Organ bath experiments, confocal microscopy and RT‐PCR were additionally used. The 5‐HT content in lung homogenates was measured by HPLC.

ResultsAntigenic challenge significantly decreased PD200, indicating the development of AI‐AHR. This hyperresponsiveness was abolished by a combination of methiothepin (5‐HT1/5‐HT2/5‐HT5/5‐HT6/5‐HT7receptors antagonist) and tropisetron (5‐HT3/5‐HT4antagonist). Other 5‐HT receptor antagonists showed three different patterns of response. Firstly, WAY100135 (5‐HT1Aantagonist) and ondansetron (5‐HT3antagonist) did not modify the AI‐AHR. Secondly, SB269970 (5‐HT7antagonist), GR113808 (5‐HT4antagonist), tropisetron or methiothepin abolished the AI‐AHR. Thirdly, ketanserin (5‐HT2Aantagonist) produced airway hyporresponsiveness. Animals with bilateral vagotomy did not develop AI‐AHR. Experiments in tracheal rings showed that pre‐incubation with LP44 or cisapride (agonists of 5‐HT7and 5‐HT4receptors, respectively) induced a significant increase of the cholinergic contractile response to the electrical field stimulation. In sensitized lung parenchyma strips, ketanserin diminished the contractile responses to ACh. Sensitization was associated with a ninefold increase in the 5‐HT content of lung homogenates. Confocal microscopy showed that sensitization enhanced the immunolabelling and co‐localization of nicotinic receptor and 5‐HT in airway epithelium, probably located in pulmonary neuroendocrine cells (PNECs). RT‐PCR demonstrated that neither sensitization nor antigen challenge modified the 5‐HT2Areceptor mRNA levels.

ConclusionsOur results suggested that 5‐HT was involved in the development of AI‐AHR to ACh in guinea‐pigs. Specifically, 5‐HT2A, 5‐HT4and 5‐HT7receptors seem to be particularly involved in this phenomenon. Participation of 5‐HT might probably be favoured by the enhancement of the PNECs 5‐HT content observed after sensitization.

Cite this as: P. Segura, M. H. Vargas, G. Córdoba‐Rodríguez, J. Chávez, J. L. Arreola, P. Campos‐Bedolla, V. Ruiz, L. M. García‐Hernández, C. Méndez and L. M. Montaño,Clinical & Experimental Allergy, 2010 (40) 327– 338.
Systemic and local anti-C5 therapy reduces the disease severity in experimental autoimmune uveoretinitis
Authors: Copland D A , Hussain K , Baalasubramanian S , Hughes T R , Morgan B P , Xu H , Dick A D , Nicholson L B
Published: December 2009

Activation of complement occurs during autoimmune retinal and intraocular inflammatory disease as well as neuroretinal degenerative disorders. The cleavage of C5 into fragments C5a and C5b is a critical event during the complement cascade. C5a is a potent proinflammatory anaphylatoxin capable of inducing cell migration, adhesion and cytokine release, while membrane attack complex C5b-9 causes cell lysis. Therapeutic approaches to prevent complement-induced inflammation include the use of blocking monoclonal antibodies (mAb) to prevent C5 cleavage. In these current experiments, the rat anti-mouse C5 mAb (BB5.1) was utilized to investigate the effects of inhibition of C5 cleavage on disease progression and severity in experimental autoimmune uveoretinitis (EAU), a model of organ-specific autoimmunity in the eye characterized by structural retinal damage mediated by infiltrating macrophages. Systemic treatment with BB5.1 results in significantly reduced disease scores compared with control groups, while local administration results in an earlier resolution of disease. In vitro, contemporaneous C5a and interferon-γ signalling enhanced nitric oxide production, accompanied by down-regulation of the inhibitory myeloid CD200 receptor, contributing to cell activation. These experiments demonstrate that C5 cleavage contributes to the full expression of EAU, and that selective C5 blockade via systemic and local routes of administration can suppress disease. This presents great therapeutic potential to protect against tissue damage during autoimmune responses in the retina or inflammation-induced degenerative disease.
Four integration patterns: a socio‐technical approach to integration in IS development projects
Authors: Bygstad Bendik , Nielsen Peter Axel , Munkvold Bjørn Erik
Published: December 2009

This paper aims to contribute to a theory of integration within the field of information systems (IS) project management. Integration is a key IS project management issue when new systems are developed and implemented into an increasingly integrated information infrastructure in corporate and governmental organizations. Expanding the perspective of traditional project management research, we draw extensively on central insights from IS research. Building on socio‐technical IS research and software engineering research, we suggest four generic patterns of integration: big bang, stakeholder integration, technical integration and socio‐technical integration. We analyse and describe the advantages and disadvantages of each pattern. The four patterns are ideal types. To explore the forces and challenges in these patterns, three longitudinal case studies were conducted. In particular we investigate the management challenges for each pattern. We find that the patterns are context‐sensitive and describe the different contexts where the patterns are applicable. For IS project management, the four integration patterns are a contribution to the management of integration risks – extending the vocabulary for assessing and mitigating these risks in IS development. For practitioners the four integration patterns represent an analytical framework to be used in planning modern IS development projects.
A comparative study of the spatial distribution of mast cells and microvessels in the foetal, adult human thymus and thymoma
Authors: Raica Marius , Cimpean Anca Maria , Nico Beatrice , Guidolin Diego , Ribatti Domenico
Published: December 2009

Mast cells (MCs) are widely distributed in human and animal tissues and have been shown to play an important role in angiogenesis in normal and pathological conditions. Few data are available about the relationship between MCs and blood vessels in the normal human thymus, and there are virtually no data about their distribution and significance in thymoma. The aim of this study was to analyse the spatial distribution of MCs and microvessels in the normal foetal and adult thymus and thymoma. Twenty biopsy specimens of human thymus, including foetal and adult normal thymus and thymoma were analysed. Double staining with CD34 and mast cell tryptase was used to count both mast cells and microvessels in the same fields. Computer‐assisted image analysis was performed to characterize the spatial distribution of MCs and blood vessels in selected specimens. Results demonstrated that MCs were localized exclusively to the medulla. Their number was significantly higher in thymoma specimens as compared with adult and foetal normal specimens respectively. In contrast the microvessel area was unchanged. The analysis of the spatial distribution and relationship between MCs and microvessels revealed that only in the thymoma specimens was there a significant spatial association between MCs and microvessels. Overall, these data suggest that MCs do not contribute significantly to the development of the vascular network in foetal and adult thymus, whereas in thymoma they show a close relationship to blood vessels. This could be an expression of their involvement not only in endothelial cells but also in tumour cell proliferation.
Increased calcium influx in the presence of ethanol in mouse pancreatic acinar cells
Authors: Del Castillo‐Vaquero Angel , Salido Ginés M. , González Antonio
Published: December 2009

The effects of alcohol on Ca2+ signalling remains poorly understood. Here we have investigated the effects of acute ethanol exposure on Ca2+ influx in mouse pancreatic acinar cells. Cells were loaded with fura‐2 and the changes in fluorescence were monitored by spectrofluorimetry and imaging analysis. Stimulation of cells with 20 pM cholecystokinin evoked an oscillatory pattern in [Ca2+]c, both in the presence and in the absence of extracellular Ca2+. Stimulation of cells with cholecystokinin in the presence of 50 mM ethanol led to a transformation of physiological oscillations into a single transient increase in [Ca2+]c. This effect was observed when Ca2+ was present in the extracellular medium, and did not appear in its absence. Addition of 1 mM CaCl2 to the extracellular medium, following release of Ca2+ from intracellular stores by stimulation of cells with 1 nM cholecystokinin or 1 μM thapsigargin in the absence of extracellular Ca2+, was followed by an increase in [Ca2+]c. Ca2+ influx was increased in the presence of 50 mM ethanol. The anti‐oxidant cinnamtannin B‐1 (10 μM) or inhibition of alcohol dehydrogenase by 4‐MP (1 mM), significantly reduced Ca2+ influx evoked by cholecystokinin in the presence of ethanol. In summary, intoxicating concentrations of ethanol may lead to over stimulation of pancreatic acinar cells by cholecystokinin. This might be partially explained by the generation of reactive oxygen species and an increased Ca2+ entry in the presence of ethanol. Potentially ethanol might lead to Ca2+ overload, which is a common pathological precursor that is implicated in pancreatitis.
Tits on the move: exploring the impact of environmental change on blue tit and great tit migration distance
Authors: Smallegange Isabel M. , Fiedler Wolfgang , Köppen Ulrich , Geiter Olaf , Bairlein Franz
Published: December 2009

1.In response to warmer spring conditions in Central Europe many migratory bird species have shifted their timing of breeding. Environmental change has also led to warmer winters, shortening the distance between the breeding grounds of migratory birds and their overwintering areas.

2.Here, we show that in response to warmer winters, blue tits (Cyanistes caeruleus), but not great tits (Parus major), breeding in Germany decreased their migration distance between 1964 and 1996. Understanding this difference provides insight into possible constraints and selection pressures involved in how species respond to environmental change. Here, we focus on their breeding ecology.

3.In a nest box population in southern Germany, both species laid their first clutch earlier with increasing spring temperature, but over the study period (1974–1999) blue tits showed a significant and stronger advancement in laying date than great tits. For both species, selection for earlier breeding did not vary with environmental change, indicating that early laying pairs did not do better than later laying pairs as spring temperature increased.

4.Blue tits in the nest box population were single‐brooded and existing hypotheses state that single‐brooded species likely advance their laying date to match timing of reproduction with the advancing food peak in spring. We hypothesize that this might be one reason why blue tits adjusted their migration strategy as closer proximity to the breeding grounds in winter allows better prediction of the onset of spring. Ten per cent of great tits successfully produced second broods and their first clutch laying date is a compromise between first and second clutch laying date, which might be why great tits had not advanced their laying date nor altered their migration strategy.
Corticotrophin‐Releasing Factor Alters the Timing of Puberty in the Female Rat
Authors: Kinsey‐Jones J. S. , Li X. F. , Knox A. M. I. , Lin Y. S. , Milligan S. R. , Lightman S. L. , O’Byrne K. T.
Published: December 2009

Puberty is a developmental process that is dependent upon activation of the hypothalamic gonadotrophin‐releasing hormone (GnRH) pulse generator. It is well established that the stress neuropeptide, corticotrophin‐releasing factor (CRF), has a profound inhibitory action on GnRH pulse generator frequency. Although stress is known to affect the timing of puberty, the role of CRF is unknown. The present study aimed to test the hypothesis that CRF plays a critical role in the timing of puberty. On postnatal day (pnd) 28, female rat pups were chronically implanted with i.c.v. cannulae and received 14 days of administration of either CRF, CRF receptor antagonist (astressin‐B) or artificial cerebrospinal fluid via an osmotic mini‐pump. A separate group of rats served as nonsurgical controls. As a marker of puberty, rats were monitored for vaginal opening and first vaginal oestrus. Levels of CRF, CRF receptor types 1 and 2 (CRF‐R1, CRF‐R2) mRNA expression in micropunches of the medial preoptic area (mPOA), hypothalamic paraventricular nucleus (PVN) and arcuate nucleus (ARC) were determined across pubertal development; brain tissue was collected from a naive group of rats on pnd 14, 32, on the day of vaginal opening, and pnd 77 (Adult). Administration of CRF resulted in a delay in the onset of puberty, whereas astressin‐B advanced pubertal onset. Additionally, CRF and CRF‐R1 mRNA expression was reduced in the mPOA, but not ARC, at puberty. In the PVN, expression of CRF, but not CRF‐R1 mRNA, was reduced at the time of puberty. These data support the hypothesis that CRF signalling may play an important role in modulating the timing of puberty in the rat.
Photoperiodic Modulation of Clock Gene Expression in the Avian Premammillary Nucleus
Authors: Leclerc B. , Kang S. W. , Mauro L. J. , Kosonsiriluk S. , Chaiseha Y. , El Halawani M. E.
Published: December 2009

The premammillary nucleus (PMM) has been shown to contain a daily endogenous dual‐oscillation in dopamine (DA)/melatonin (MEL) as well as c‐fos mRNA expression that is associated with the daily photo‐inducible phase of gonad growth in turkeys. In the present study, the expression of clock genes (Bmal1, Clock, Cry1, Cry2, Per2 and Per3) in the PMM was determined under short (8 : 16 h light/dark cycle) and long (16 : 8 h light/dark cycle) photoperiods relative to changes associated with the diurnal rhythm of DA and MEL. Constant darkness (0 : 24 h light/dark cycle) was used to assess the endogenous response of clock genes. In addition, light pulses were given at zeitgeber time (ZT) 8, 14 and 20 to ascertain whether clock gene expression is modulated by light pulse stimulation and therefore has a daily phase‐related response. In the PMM, the temporal clock gene expression profiles were similar under short and long photoperiods, except that Per3 gene was phase‐delayed by approximately 16 h under long photoperiod. In addition, Cry1 and Per3 genes were light‐induced at ZT 14, the photosensitive phase for gonad recrudescence, whereas the Clock gene was repressed. Gene expression in established circadian pacemakers, the visual suprachiasmatic nucleus (vSCN) and the pineal, was also determined. Clock genes in the pineal gland were rhythmic under both photoperiods, and were not altered after light pulses at ZT 14, which suggests that pineal clock genes may not be associated with the photosensitive phase and reproductive activities. In the vSCN, clock gene expression was phase‐shifted depending on the photoperiod, with apexes at night under short day length and during the day under long day length. Furthermore, light pulses at ZT 14 induced the Per2 gene, whereas it repressed the Bmal1 gene. Taken together, the changes in clock gene expression observed within the PMM were unique compared to the pineal and vSCN, and were induced by long photoperiod and light during the daily photosensitive phase; stimuli that are also documented to promote reproductive activity. These results show that Cry1 and Per3 are involved in the photic response associated with the PMM neuronal activation and are coincident with an essential circadian mechanism (photosensitive phase) controlling the reproductive neuroendocrine system.
Host specificity, phenotype matching and the evolution of reproductive isolation in a coevolved plant–pollinator mutualism
Authors: HIMLER ANNA G. , MACHADO CARLOS A.
Published: December 2009

Coevolutionary interactions between plants and their associated pollinators and seed dispersers are thought to have promoted the diversification of flowering plants (Raven 1977; Regal 1977; Stebbins 1981). The actual mechanisms by which pollinators could drive species diversification in plants are not fully understood. However, it is thought that pollinator host specialization can influence the evolution of reproductive isolation among plant populations because the pollinator’s choice of host is what determines patterns of gene flow in its host plant, and host choice may also have important consequences on pollinator and host fitness (Grant 1949; Bawa 1992). In this issue of Molecular Ecology, Smith et al. (2009) present a very interesting study that addresses how host specialization affects pollinator fitness and patterns of gene flow in a plant host. Several aspects of this study match elements of a seminal mathematical model of plant–pollinator codivergence (Kiester et al. 1984) suggesting that reciprocal selection for matched plant and pollinator reproductive traits may lead to speciation in the host and its pollinator when there is strong host specialization and a pattern of geographic subdivision. Smith et al.’s study represents an important step to fill the gap in our understanding of how reciprocal selection may lead to speciation in coevolved plant–pollinator mutualisms.
Methanogenesis by Methanosarcina acetivorans involves two structurally and functionally distinct classes of heterodisulfide reductase
Authors: Buan Nicole R. , Metcalf William W.
Published: December 2009

Biochemical studies have revealed two distinct classes of Coenzyme B‐Coenzyme M heterodisulfide (CoB‐S‐S‐CoM) reductase (Hdr), a key enzyme required for anaerobic respiration in methane‐producing archaea. A cytoplasmic HdrABC enzyme complex is found in most methanogens, whereas a membrane‐bound HdrED complex is found exclusively in members of the order Methanosarcinales. Unexpectedly, genomic data indicate that multiple copies of both Hdr classes are found in all sequenced Methanosarcinales genomes. The Methanosarcina acetivorans hdrED1 operon is constitutively expressed and required for viability under all growth conditions examined, consistent with HdrED being the primary Hdr. HdrABC appears to be specifically involved in methylotrophic methanogenesis, based on reduced growth and methanogenesis rates of an hdrA1C1B1 mutant on methylotrophic substrates and downregulation of the genes during growth on acetate. This conclusion is further supported by phylogenetic analysis showing that the presence of hdrA1 in an organism is specifically correlated with the presence of genes for methylotrophic methanogenesis. Examination of mRNA abundance in methanol‐grown ΔhdrA1C1B1 strains relative to wild‐type revealed upregulation of genes required for synthesis of (di)methylsulfide and for transport and biosynthesis of CoB‐SH and CoM‐SH, suggesting that the mutant has a defect in electron transfer from ferredoxin to CoB‐S‐S‐CoM that causes cofactor limitation.
Sequential translocation of an Escherchia coli two‐partner secretion pathway exoprotein across the inner and outer membranes
Authors: Choi Peter S. , Bernstein Harris D.
Published: December 2009

In Gram‐negative bacteria, a variety of high molecular weight ‘exoproteins’ are translocated across the outer membrane (OM) via the two‐partner secretion (TPS) pathway by interacting with a dedicated transporter. It is unclear, however, whether the translocation of exoproteins across the OM is coupled to their translocation across the inner membrane (IM). To address this question, we separated the production of an Escherichia coli O157:H7 exoprotein (OtpA) and its transporter (OtpB) temporally by placing otpA and otpB under the control of distinct regulatable promoters. We found that when both full‐length and truncated forms of OtpA were expressed prior to OtpB, a significant fraction of the exoprotein was secreted. The results indicate that OtpA can be translocated into the periplasm and briefly remain secretion‐competent. Furthermore, by engineering cysteine residues into OtpA and using disulphide bond formation as a reporter of periplasmic localization, we obtained additional evidence that the C‐terminus of OtpA enters the periplasm before the N‐terminus is translocated across the OM even when OtpA and OtpB are expressed simultaneously. Taken together, our results demonstrate that the translocation of a TPS exoprotein across the OM can occur independently from its translocation across the IM.
Differential effects and interactions of endogenous and horizontally acquired H‐NS‐like proteins in pathogenic Escherichia coli
Authors: Müller Claudia M. , Schneider György , Dobrindt Ulrich , Emödy Levente , Hacker Jörg , Uhlin Bernt Eric
Published: December 2009

The nucleoid‐associated protein H‐NS is important for gene regulation in Escherichia coli. We have studied H‐NS interaction with StpA and an uncharacterized H‐NS‐like protein, Hfp, in the uropathogenic E. coli isolate 536 that expresses all three nucleoid‐associated proteins. We found distinct interactions of the three proteins at the protein level, resulting in the formation of heteromers, as well as differences in their gene expression at the transcriptional level. Mutants lacking either StpA or Hfp alone did not exhibit a phenotype at 37°C, which is consistent with a low level of expression at that temperature. Expression of the hfp and stpA genes was found to be induced by apparently diametrical conditions, and StpA and Hfp levels could be correlated to modulatory effects on the expression of different H‐NS targets, the bgl operon and operons for virulence factors such as fimbriae and capsular polysaccharide. The hns/hfp and hns/stpA double mutants displayed severe growth defects at low and high temperatures respectively. Our findings demonstrated different requirements for the alternative H‐NS/Hfp/StpA combinations under these growth conditions. We propose that Hfp and StpA have distinct functions and roles in a dynamic pool of nucleoid‐associated proteins that is adapting to requirements in a particular environment.
Transcriptional regulation of the Yts1 type II secretion system of Yersinia enterocolitica and identification of secretion substrates
Authors: Shutinoski Bojan , Schmidt M. Alexander , Heusipp Gerhard
Published: December 2009

Type II secretion systems (T2SSs) mediate the transport of proteins through the bacterial outer membrane. Although widespread in γ‐proteobacteria, they have so far not been characterized in the human pathogen Yersinia enterocolitica. We describe here genetic linkage of the Yts1 and Yts2 T2SSs with a transcriptional regulator in the highly pathogenic Y. enterocolitica biotype 1B. While the Yts2‐associated PypC regulator activates the Yts2 operon, the PclR regulator does not induce transcription of its cognate Yts1 operon. Instead, Yts1 and pclR are activated by the addition of MgCl2 to the growth medium at 17°C and 26°C, but not at 37°C. We identified three proteins, ChiY, EngY (YE2830) and YE3650, that are secreted depending on a functional Yts1 T2SS in response to MgCl2 at low temperature. While the activation of chiY by MgCl2 depends on pclR, PclR overproduction is not sufficient for chiY transcription in an Escherichia coli background, demonstrating the need for additional Y. enterocolitica‐specific factors. As ChiY and EngY both bind to chitin, and YE3650 shows motifs conserved in oligosaccharide‐binding enzymes, all secreted proteins might be important for polysaccharide interaction/degradation during infection or in the environment.
Ordered association of helicase loader proteins with the Bacillus subtilis origin of replication in vivo
Authors: Smits Wiep Klaas , Goranov Alexi I. , Grossman Alan D.
Published: December 2009

The essential proteins DnaB, DnaD and DnaI of Bacillus subtilis are required for initiation, but not elongation, of DNA replication, and for replication restart at stalled forks. The interactions and functions of these proteins have largely been determined in vitro based on their roles in replication restart. During replication initiation in vivo, it is not known if these proteins, and the replication initiator DnaA, associate with oriC independently of each other by virtue of their DNA binding activities, as a (sub)complex like other loader proteins, or in a particular dependent order. We used temperature‐sensitive mutants or a conditional degradation system to inactivate each protein and test for association of the other proteins with oriC in vivo. We found that there was a clear order of stable association with oriC; DnaA, DnaD, DnaB, and finally DnaI‐mediated loading of helicase. The loading of helicase via stable intermediates resembles that of eukaryotes and the established hierarchy provides several potential regulatory points. The general approach described here can be used to analyse assembly of other complexes.
National health insurance coverage and socio‐economic status in a rural district of Ghana
Authors: Sarpong N. , Loag W. , Fobil J. , Meyer C. G. , Adu‐Sarkodie Y. , May J. , Schwarz N. G.
Published: December 2009

Objective  To explore the association between socio‐economic status (SES) and health insurance subscription to the Ghanaian National Health Insurance Scheme (NHIS) of residents of the Asante Akim North district of the Ashanti Region, Ghana.

Methods  In the course of a community survey, data on asset variables (e.g. electricity, housing conditions and other variables) and on NHIS subscription were collected on the household level in 99 villages. Using principal components analysis, households were classified into three categories of SES (20% high, 40% middle and 40% low SES). Odds ratios of NHIS subscription were calculated for all SES categories, using the low category as the reference group and adjusting for travelling time to health facilities by public transport.

Results  Of the 7223 households surveyed, 38% subscribed to the NHIS, of these 21% were low, 43% middle and 60% high SES households. SES was significantly associated with NHIS subscription (high SES: OR 4.9, 95% CI 4.3–5.7; middle SES: OR 2.5, 95% CI 2.2–2.9; low SES: OR 1, reference group).

Conclusion  Four years after its introduction, the NHIS has reached subscription rates of 38% in the district surveyed. However, to achieve the aim of assuring universal access to health care facilities for all residents of Ghana, in particular for individuals living under socio‐economic constraints, increasing subscription rates are necessary.
Blood glucose and prognosis in children with presumed severe malaria: is there a threshold for ‘hypoglycaemia’?
Authors: Willcox Merlin L. , Forster Mathieu , Dicko Moussa I. , Graz Bertrand , Mayon‐White Richard , Barennes Hubert
Published: December 2009

Objectives  Hypoglycaemia (glucose <2.2 mmol/l) is a defining feature of severe malaria, but the significance of other levels of blood glucose has not previously been studied in children with severe malaria.

Methods  A prospective study of 437 consecutive children with presumed severe malaria was conducted in Mali. We defined hypoglycaemia as <2.2 mmol/l, low glycaemia as 2.2–4.4 mmol/l and hyperglycaemia as >8.3 mmol/l. Associations between glycaemia and case fatality were analysed for 418 children using logistic regression models and a receiver operator curve (ROC).

Results  There was a significant difference between blood glucose levels in children who died (median 4.6 mmol/l) and survivors (median 7.6 mmol/l, P < 0.001). Case fatality declined from 61.5% of the hypoglycaemic children to 46.2% of those with low glycaemia, 13.4% of those with normal glycaemia and 7.6% of those with hyperglycaemia (P < 0.001). Logistic regression showed an adjusted odds ratio (AOR) of 0.75 (0.64–0.88) for case fatality per 1 mmol/l increase in baseline blood glucose. Compared to a normal blood glucose, hypoglycaemia and low glycaemia both significantly increased the odds of death (AOR 11.87, 2.10–67.00; and 5.21, 1.86–14.63, respectively), whereas hyperglycaemia reduced the odds of death (AOR 0.34, 0.13–0.91). The ROC [area under the curve at 0.753 (95% CI 0.684–0.820)] indicated that glycaemia had a moderate predictive value for death and identified an optimal threshold at glycaemia <6.1 mmol/l, (sensitivity 64.5% and specificity 75.1%).

Conclusions  If there is a threshold of blood glucose which defines a worse prognosis, it is at a higher level than the current definition of 2.2 mmol/l.