bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrasonic nebulization of hypertonic solution: a new method for obtaining specimens from nasal mucosa for morphologic and biochemical analysis in allergic rhinitis
Authors: Melillo G. , Balzano G. , Stefanelli F. , Iorio C. , De Angelis E. , Melillo E.
Published: August 2010

Various techniques are used to collect specimens from the nasal mucosa for morphologic and biochemical analysis. The purpose of this study was to devise a method that overcomes some of the disadvantages (e.g., invasive procedure, samples not suitable for cytologic and biochemical analysis, lack of standardization, and poor reproducibility) of these techniques. The new method requires subjects, with neck extended, to inhale an ultrasonic nebulization of a hypertonic (3% NaCl) solution (UNHS) for 5 min. They then blow their nose into a Petri dish, one nostril at a time with the other one blocked. The secretions are dispersed with 0.1% dithiothreitol in phosphate buffer solution for 20 min. Total cell count (TCC) is evaluated, and the cellular suspension is divided into two aliquots: one is centrifuged and the supernatants are collected for eosinophil cationic protein (ECP) measurements; the other is cytocentrifuged and the slides, stained with Diff‐Quik, are used for differential cell count. The results obtained with the UNHS and nasal lavage (NL) methods were compared. Eleven nonatopic healthy subjects and 19 allergic rhinitic patients were studied. Total cell count (×105) was significantly higher with UNHS than with NL (13.0±12.3 vs 1.911.6; P<0.0]) The differential cell count was similar with the two procedures. ECP levels (μg/l) were higher with UNHS than with NL (39.1+38.2 V.S 16.7±41.2; P<0.01). For evaluation of reproducibility, four healthy and six rhinitic subjects underwent UNHS on two occasions within 5 days, and the results of two samples (sample 1 vs sample 2) were analyzed. Reproducibility was good as to TCC, differential cell count, and ECP
The association between serum levels of Th cytokines and rhinoconjunctivitis caused by methyltetrahydrophthalic anhydride
Authors: Yokot K. , Yamaguchi K. , Takeshita T. , Morimoto K.
Published: August 2010

Background There are few epidemiologic studies on the association between serum levels of T helper (Th) cytokines and allergic symptoms caused by sensitizing agents.

Methods A population of 147 workers from two condenser plants using epoxy resin with methyltetrahydrophthalic anhydride (MTHPA) underwent a questionnaire survey and serologic investigations. Total and MTHPA‐specific IgE levels were measured by the Pharmacia CAP System, and serum levels of interleukin (IL)‐4, IL‐13, and interferon‐gamma (IFN‐γ) by enzyme immunoassay.

Results A significant association was found between work‐related eye and nasal symptoms and a range of atopic characteristics, including the frequency of positive specific IgE, specific and total IgE levels, and IL‐4 levels. A positive correlation was observed between levels of IL‐4 and IL‐13 (r=0.42). Furthermore, multiple logistic analysis revealed significant contributions of specific IgE and I H3 to the work‐related symptoms. A similar but nonsignificant association was also found for IL‐4 (P=0.07). On the other hand, a multiple regression model with specific IgE levels as a dependent variable showed a significant association with total IgE, but not with IL‐4, IL‐13, and IFN‐γ.

Conclusions These results suggest that work‐related eye and nasal symptoms are closely related to specific IgE antibodies, and that allergic responses, mediated by IgE, to MTHPA may lead to a shift in the balance between Thl and Th2 cells.
Reactivity to recombinant house‐dust‐mite allergens in asthma and rhinitis in a tropical environment
Authors: Lynch N. R. , Puccio F. A. , Di Prisco M. C. , Lopez R. I. , Hazell L. A. , Smith W. A. , Thomas W. R.
Published: August 2010

Background House‐dust mites contain components that are allergenic in mite‐sensitive patients, and a number of these have been produced in recombinant form.

Methods In the present study, we evaluated by skin prick testing the positivity to native Der p 2 and recombinant Der p 2, Der p 5, and Der p 7 allergens of Dermatophagoides pteronyssinus in patients with rhinitis, asthma, or a combination of these diseases, who were positive to whole‐mite extract. Results In all patients, the positivity to both native and recombinant Der 2 was high. In patients with either rhinitis or asthma, the reactivity to Der p 5 and 7 was significantly lower than to Der p 2. However, in patients with combined disease, the positivity to the minor allergens was almost as high as that to Der p 2.

Conclusions These results raise the question of whether patients with combined allergic rhinitis and asthma, when compared to those with either of these diseases alone, are predisposed to react to a wider range of mite allergens, or, inversely, whether patients who respond to the minor allergens are more susceptible to suffering the combined disease.
Desloratadine activity in concurrent seasonal allergic rhinitis and asthma
Authors: Baena‐Cagnani C. E.
Published: August 2010

Seasonal allergic rhinitis (SAR) and asthma, which are frequently comorbid, share some common allergic pathogenic bases. Clinical manifestations of these disorders might therefore be viewed as local manifestations of a systemic inflammatory state. Not only do the onsets of allergic‐rhinitis (AR) and asthma symptoms often coincide (within 1 year), but also nasal challenges with SAR allergens can induce airways hyperreactivity (AHR). Eosinophils, which are key effector cells in both SAR and asthma, cause AHR, tissue damage, and neuronal effects through secretion of toxic granule proteins, enzymes, and other mediators. The novel, nonsedating, histamine H1‐receptor antagonist, desloratadine, which exerts various favorable effects on the allergic cascade, significantly decreased SAR symptoms (e.g., nasal congestion) and diminished daily β2‐agonist use and improved asthma symptoms, while maintaining pulmonary function, in patients with SAR‐asthma who were treated with once‐daily desloratadine regimens.
Nitrogen washout slope in poorly controlled asthma
Authors: Bourdin A. , Paganin F. , Préfaut C. , Kieseler D. , Godard P. , Chanez P.
Published: August 2010

Short‐term control of asthma is often lacking even though forced expiratory volume in 1 s (FEV1) remains above normal value. Small airways are a potential key site of persistent inflammation and structural modification. Noninvasive assessment of small airways was found to be difficult, but the computerized single breath nitrogen washout test (SBNT) has been recently successfully reintroduced with this aim. Twenty‐four asthmatics (13 females) of various severity but with normal FEV1 were compared with 24 healthy volunteers (13 females) and studied at steady‐state after bronchodilatation (400 μg salbutamol). Spirometric values, plethysmographic data, phase III (slope of phase III of the SBNT, dN2) and IV [closing volume (CV), with closing capacity (CC) = CV + residual volume (RV)] of the SBNT were checked. Asthma severity, recent control, exacerbation rate, and therapy requirements were assessed on the basis of validated questionnaires (ACQ) and international guidelines. Patients were prospectively pooled into two equal groups according to their exacerbation rate. The reproducibility of the measurements obtained on 2 following days was assessed. All plethysmographic values, except total lung capacity (TLC), differentiated asthmatic patients from controls. The CC/TLC [124 (117–148) vs 117 (112–123), P = 0.04] and dN2 [110 (99–190) vs 94 (75–111), P = 0.02] were increased in asthma. The dN2 was significantly increased in patients with frequent exacerbations [100 (83–105) vs 195 (141–212), P = 0.0005]. A correlation was obtained between dN2 and recent asthma control (ρ: 0.62; P = 0.003), number of exacerbations (ρ: 0.71, P = 0.0008), and RV/TLC (ρ: 0.49, P = 0.026). This study demonstrated that ventilation inequalities assessed by dN2 represent an important indicator of poor asthma control and high exacerbation rate in high symptom perceivers. New therapies focused on small airways should now be developed.
Screening the allergenic repertoires of wheat and maize with sera from double‐blind, placebo‐controlled food challenge positive patients
Authors: Weichel M. , Vergoossen N. J. , Bonomi S. , Scibilia J. , Ortolani C. , Ballmer‐Weber B. K. , Pastorello E. A. , Crameri R.
Published: August 2010

Background:  Food allergy to wheat and maize is an increasing factor of deterioration of life quality, especially childhood and can, in rare cases, even induce anaphylaxis. Although omega‐5 gliadin from wheat and maize lipid transfer protein have been characterized as major cereal allergens on the molecular level, the list of food allergens is far to be complete.

Methods:  To identify the IgE‐binding repertoires of wheat and maize we screened respective cDNA libraries displayed on phage surface with sera from patients with a confirmed food allergy. The study included six patients with a positive double‐blind, placebo‐controlled food challenge (DBPCFC) to wheat, nine patients with a positive DBPCFC to maize, and six patients with anaphylactic reactions after ingestion of wheat.

Results:  The enriched sequences encoding IgE‐binding proteins showed heterogeneous repertoires for both, wheat and maize. The selected wheat repertoire yielded 12, the maize repertoire 11 open reading frames. Among these we identified allergens belonging to already characterized allergens families, such as gliadin, profilin and beta‐expansin. Besides, we found novel proteins with high cross‐reactive potential, such as thioredoxins, as well as sequences that had so far not been related to cereal allergy at all. The IgE‐binding capacity of some selected proteins was evaluated in vitro and cross‐reactivity was demonstrated by competition ELISA.

Conclusion:  With regard to the heterogeneity of the characterized sequences as well as to the biochemical nature of the new allergens detected we conclude that wheat and maize‐related food allergy is more complex than so far anticipated.
Role of cysteinyl leukotrienes in human allergen‐specific Th2 responses induced by granulocyte macrophage‐colony stimulating factor
Authors: Faith A. , Fernandez M. H. , Caulfield J. , Loke T.‐K. , Corrigan C. , O’Connor B. , Lee T. H. , Hawrylowicz C. M.
Published: August 2010

Background:  The pro‐inflammatory cytokine, granulocyte macrophage‐colony stimulating factor (GM‐CSF), which is elevated in the lungs of atopic asthmatic patients, has been shown to enhance major histocompatibility class II expression of alveolar macrophages (AM). We hypothesized that exposure of AM and monocytes from atopic asthmatic patients to GM‐CSF would enhance their antigen presenting function, and investigated putative mechanisms for this effect.

Methods:  Alveolar macrophages were purified from bronchoalveolar lavage by plastic adherence. Monocytes and CD4+ T cells were purified from peripheral blood by magnetic bead separation. Antigen‐presenting cell (APC) were pretreated with GM‐CSF, pulsed with allergen and cocultured with autologous T cells. T‐cell proliferation was determined by tritiated thymidine incorporation and cytokine production by enzyme‐linked immunosorbent assay.

Results:  Exposure of allergen‐pulsed AM and peripheral blood monocytes to GM‐CSF significantly increased allergen‐specific T‐cell proliferation and T helper 2 (Th2) cytokine production. The enhanced response was dependent on costimulation by CD86, but not CD80. Inhibition of the 5‐lipoxygenase pathway abrogated GM‐CSF‐mediated upregulation by monocytes of allergen‐specific interleukin‐5 (IL‐5) and IL‐13 cytokine production. Blocking of the cysteinyl leukotriene receptor 1 (cysLT1) receptor by a specific receptor antagonist inhibited allergen‐specific IL‐5 production in response to GM‐CSF pretreatment.

Conclusion:  Exposure to GM‐CSF enhanced the capacity of human APC from atopic asthmatic patients to induce allergen‐specific Th2 responses by a mechanism involving cysLT. Novel immunotherapies, targeting production of GM‐CSF or its actions on APC have the potential, therefore, to prove beneficial in treatment of patients with inflammatory airway disease.
Tolerability of meropenem in children with IgE‐mediated hypersensitivity to penicillins
Authors: Atanasković‐Marković M. , Gaeta F. , Medjo B. , Viola M. , Nestorović B. , Romano A.
Published: August 2010

Background: Administration of meropenem to penicillin‐allergic patients who might benefit from this treatment is usually avoided because of a 47.4% rate of cross‐reactivity to imipenem, the prototype of the carbapenem class of β‐lactam antibiotics, demonstrated in a single study on the basis of positive responses to skin tests with imipenem reagents. However, recent studies of ours have demonstrated a very low rate of cross‐reactivity between penicillins and both meropenem and imipenem in adults.

Objective: To assess cross‐reactivity and tolerability of meropenem in children with documented penicillin allergy.

Methods: One hundred and eight consecutive children who had suffered a total of 129 immediate reactions (120 urticarial and/or angioedematous manifestations and 9 anaphylactic shocks) to penicillins and had positive results to skin tests for at least one of the penicillin reagents tested underwent skin tests with meropenem and negative subjects were challenged with it.

Results: One subject (0.9%) displayed a positive intradermal test to meropenem. The remaining 107 subjects with negative skin tests to meropenem tolerated challenges. Challenges were not followed by full therapeutic courses.

Conclusions: Our results demonstrate a low rate of cross‐reactivity between penicillins and meropenem. Therefore, the practice of avoiding meropenem in children with immunoglobulin E‐mediated hypersensitivity could be abandoned; in those who especially require meropenem treatment, prophylactic skin tests are advisable, because negative results indicate tolerability.
Methodology for development of the Allergic Rhinitis and its Impact on Asthma Guideline 2008 update
Authors: Brozek J. L. , Baena‐Cagnani C. E. , Bonini S. , Canonica G. W. , Rasi G. , Van Wijk R. G. , Zuberbier T. , Guyatt G. , Bousquet J. , Schünemann H. J.
Published: August 2010

Background:  We describe the methodology for the 2008 update of the Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines. The methodology differs from the 2001 edition in several respects. The most prominent change is the application of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach to compiling evidence, assessing the quality of evidence and grading of recommendations.

Methods and results:  Representatives of the GRADE working group joined the ARIA guideline panel to achieve these tasks. While most recommendations result from existing systematic reviews, systematic reviews were not always available and the panel compiled the best available evidence in evidence profiles without conducting actual reviews. The panel conducted two meetings and used the GRADE criteria to assess the quality of evidence (four categories of high, moderate, low and very low) and the strength of recommendation (strong and weak) based on weighing up the desirable and undesirable effects of management strategies, considering values and preferences influencing recommendations, and resource implications. The guideline panel has chosen the words ‘we recommend’– for strong recommendations and ‘we suggest’– for weak recommendations. Both categories indicate the best course of action for a given patient population, but their implementation, requires different considerations as we describe subsequently in this article.

Conclusions:  The 2008 update of the ARIA guidelines has become more evidence‐based. Future iterations of the guidelines will further be improved by following the described processes even closer, such as ensuring availability of updated high quality systematic reviews for each question.
Novel sequences and epitopes of diagnostic value derived from the Anisakis simplex Ani s 7 major allergen*
Authors: Rodríguez E. , Anadón A. M. , García‐Bodas E. , Romarís F. , Iglesias R. , Gárate T. , Ubeira F. M.
Published: August 2010

Background:  Anisakis simplex allergens may cause severe allergic reactions in infected patients. Human anisakiasis can be specifically diagnosed by detection of immunoglobulin E (IgE) antibodies against O‐deglycosylated nAni s 7 allergen captured by monoclonal antibody (mAb) UA3 (UA3‐ELISA), although the nature of this important allergen is unknown. The aim of this study was to clone and characterize the Ani s 7 major allergen, and to obtain a recombinant fragment suitable for serodiagnosis.

Methods:  An Anisakis cDNA library was screened with mAb UA3 and a cDNA clone (rAni s 7) encoding a 1096‐amino acid fragment of Ani s 7 (GenBank: EF158010) was identified. Bioinformatic tools and immunological and biochemical techniques were used to characterize the allergen obtained.

Results:  The rAni s 7 fragment comprised 19 repeats of a novel CX17−25CX9−22CX8CX6 tandem repeat motif not seen in any previously reported protein sequence. An internal 435Met–713Arg fragment of the rAni s 7 (t‐Ani s 7) was expressed in Escherichia coli and evaluated for serodiagnostic utility. Indirect enzyme‐linked immunosorbent assay (ELISA) with t‐Ani s 7 identified as positive the same 60 sera as UA3‐ELISA. The sequence MCQCVQKYGTEFCKKRLA from rAni s 7 was identified as the epitope recognized by mAb UA3, and is the target for over 60% of human IgE antibodies that react with O‐deglycosylated nAni s 7.

Conclusions:  In addition to their clear value for serodiagnosis of human anisakiasis, the nature of the novel sequences and epitopes identified in the Ani s 7 allergen are of interest for a better understanding of the mechanisms operating in Anisakis‐induced allergy.
Anaphylaxis in patients with mastocytosis: a study on history, clinical features and risk factors in 120 patients
Authors: Brockow K. , Jofer C. , Behrendt H. , Ring J.
Published: August 2010

Background:  Excessive mast cell mediator release may lead to anaphylaxis in patients with mastocytosis. However, the incidence, clinical features and trigger factors have not yet been analyzed.

Methods:  To identify risk factors for anaphylaxis in mastocytosis, we determined cumulative incidence, severity, clinical characteristics, and trigger factors for anaphylaxis in 120 consecutive patients (53 male; 67 female, median age and range 24 years, 1 month to 73 years), and correlated these with disease severity of mastocytosis, skin involvement, basal total serum tryptase, and diaminooxidase concentrations.

Results:  The cumulative incidence of anaphylaxis in patients with mastocytosis was higher in adults (49%; P < 0.01) compared with that in children (9%). Only children with extensive skin involvement had experienced anaphylaxis. In adults, anaphylaxis was correlated to the absence of urticaria pigmentosa lesions (P < 0.03). Reactions occurred more frequently in adults with systemic (56%) when compared with cutaneous mastocytosis (13%; P < 0.02). In adults, 48% of reactions were severe, and 38% resulted in unconsciousness. Major perceived trigger factors for adults were hymenoptera stings (19%), foods (16%), and medication (9%); however, in 26% of reactions, only a combination of different triggers preceded anaphylaxis. Trigger factors remained unidentified in 67% of reactions in children compared with 13% in adults. Patients with anaphylaxis had higher basal tryptase values (60.2 ± 55 ng/ml, P < 0.0001) in comparison with those without (21.2 ± 33 ng/ml), but not diaminooxidase levels.

Conclusion:  Adult patients and children with extensive skin disease with mastocytosis have an increased risk to develop severe anaphylaxis; thus, an emergency set of medication including epinephrine is recommended.
Practical allergy (PRACTALL) report: risk assessment in anaphylaxis
Authors: Simons F. E. R. , Frew A. J. , Ansotegui I. J. , Bochner B. S. , Golden D. B. K. , Finkelman F. D. , Leung D. Y. M. , Lotvall J. , Marone G. , Metcalfe D. D. , Müller U. , Rosenwasser L. J. , Sampson H. A. , Schwartz L. B. , Van Hage M. , Walls A. F.
Published: August 2010

Effector mechanisms in anaphylaxis were reviewed. Current approaches to confirmation of the clinical diagnosis were discussed. Improved methods for distinguishing between allergen sensitization (which is common in the general population) and clinical risk of anaphylaxis (which is uncommon) were deliberated. Innovative techniques that will improve risk assessment in anaphylaxis in the future were described.
Male‐induced costs of mating for females compensated by offspring viability benefits in an insect
Authors: GARCIA‐GONZALEZ F. , SIMMONS L. W.
Published: August 2010

Sexual conflict facilitates the evolution of traits that increase the reproductive success of males at the expense of components of female fitness. Theory suggests that indirect benefits are unlikely to offset the direct costs to females from antagonistic male adaptations, but empirical studies examining the net fitness pay‐offs of the interaction between the sexes are scarce. Here, we investigate whether matings with males that invest intrinsically more into accessory gland tissue undermine female lifetime reproductive success (LRS) in the cricketTeleogryllus oceanicus. We found that females incur a longevity cost of mating that is proportional to the partner’s absolute investment into the production of accessory gland products. However, male accessory gland weight positively influences embryo survival, and harmful ejaculate‐induced effects are cancelled out when these are put in the context of female LRS. The direct costs of mating with males that sire offspring with higher viability are thus compensated by direct and possibly indirect genetic benefits in this species.
Sex‐related variation in migration phenology in relation to sexual dimorphism: a test of competing hypotheses for the evolution of protandry
Authors: SAINO N. , RUBOLINI D. , SERRA L. , CAPRIOLI M. , MORGANTI M. , AMBROSINI R. , SPINA F.
Published: August 2010

Timing of arrival/emergence to the breeding grounds is under contrasting natural and sexual selection pressures. Because of differences in sex roles and physiology, the balance between these pressures on either sex may differ, leading to earlier male (protandry) or female (protogyny) arrival. We test several competing hypotheses for the evolution of protandry using migration data for 22 bird species, including for the first time several monochromatic ones where sexual selection is supposedly less intense. Across species, protandry positively covaried with sexual size dimorphism but not with dichromatism. Within species, there was weak evidence that males migrate earlier because, being larger, they are less susceptible to adverse conditions. Our results do not support the ‘rank advantage’ and the ‘differential susceptibility’ hypotheses, nor the ‘mate opportunity’ hypothesis, which predicts covariation of protandry with dichromatism. Conversely, they are compatible with ‘mate choice’ arguments, whereby females use condition‐dependent arrival date to assess mate quality.
Geographic variation in phenotypic plasticity in response to dissolved oxygen in an African cichlid fish
Authors: CRISPO E. , CHAPMAN L. J.
Published: August 2010

Genetic adaptation and phenotypic plasticity are two ways in which organisms can adapt to local environmental conditions. We examined genetic and plastic variation in gill and brain size among swamp (low oxygen; hypoxic) and river (normal oxygen; normoxic) populations of an African cichlid fish,Pseudocrenilabrus multicolor victoriae. Larger gills and smaller brains should be advantageous when oxygen is low, and we hypothesized that the relative contribution of local genetic adaptation vs. phenotypic plasticity should be related to potential for dispersal between environments (because of gene flow’s constraint on local genetic adaptation). We conducted a laboratory‐rearing experiment, with broods from multiple populations raised under high‐oxygen and low‐oxygen conditions. We found that most of the variation in gill size was because of plasticity. However, both plastic and genetic effects on brain mass were detected, as were genetic effects on brain massplasticity. F1offspring from populations with the highest potential for dispersal between environments had characteristically smaller and more plastic brains. This phenotypic pattern might be adaptive in the face of gene flow, if smaller brains and increased plasticity confer higher average fitness across environment types.
Historical mutation rates predict susceptibility to radiation in Chernobyl birds
Authors: MØLLER A. P. , ERRITZøE J. , KARADAS F. , MOUSSEAU T. A.
Published: August 2010

Extreme environmental perturbations are rare, but may have important evolutionary consequences. Responses to current perturbations may provide important information about the ability of living organisms to cope with similar conditions in the evolutionary past. Radioactive contamination from Chernobyl constitutes one such extreme perturbation, with significant but highly variable impact on local population density and mutation rates of different species of animals and plants. We explicitly tested the hypothesis that species with strong impacts of radiation on abundance were those with high rates of historical mutation accumulation as reflected by cytochrome b mitochondrial DNA base‐pair substitution rates during past environmental perturbations. Using a dataset of 32 species of birds, we show higher historical mitochondrial substitution rates in species with the strongest negative impact of local levels of radiation on local population density. These effects were robust to different estimates of impact of radiation on abundance, weighting of estimates of abundance by sample size, statistical control for similarity in the response among species because of common phylogenetic descent, and effects of population size and longevity. Therefore, species that respond strongly to the impact of radiation from Chernobyl are also the species that in the past have been most susceptible to factors that have caused high substitution rates in mitochondrial DNA.
Ploidy mosaicism in well‐developed nuclear transplants produced by transfer of adult somatic cell nuclei to nonenucleated eggs of medaka (Oryzias latipes)
Authors: Kaftanovskaya Elena , Motosugi Nami , Kinoshita Masato , Ozato Kenjiro , Wakamatsu Yuko
Published: August 2010

Chromosomal abnormalities such as ploidy mosaicism have constituted a major obstacle to the successful nuclear transfer of adult somatic cell nuclei in lower vertebrates to date. Euploid mosaicism has been reported previously in well‐developed amphibian transplants. Here, we investigated ploidy mosaicisms in well‐developed transplants of adult somatic cell nuclei in medaka fish (Oryzias latipes). Donor nuclei from primary cultured cells from the adult caudal fin of a transgenic strain carrying the green fluorescent protein gene (GFP) were transferred to recipient nonenucleated eggs of a wild‐type strain to produce 662 transplants. While some of the transplants developed beyond the body formation stage and several hatched, all exhibited varying degrees of abnormal morphology, limited growth and subsequent death. Twenty‐one transplants, 19 embryos and two larvae, were selected for chromosomal analysis; all were well‐developed 6‐day‐old or later embryonic stages exhibiting slight morphological abnormalities and the same pattern of GFP expression as that of the donor strain. In addition, all exhibited various levels of euploid mosaicism with haploid‐diploid, haploid‐triploid or haploid‐diploid‐triploid chromosome sets. No visible chromosomal abnormalities were observed. Thus, euploid mosaicism similar to that observed in amphibians was confirmed in well‐developed nuclear transplants of fish.
Role of Bone morphogenetic protein 4 in zebrafish semicircular canal development
Authors: Omata Yasuhiro , Nojima Yasuhiro , Nakayama Satomi , Okamoto Hitoshi , Nakamura Harukazu , Funahashi Jun–ichi
Published: August 2010

Bone morphogenetic proteins (BMPs) are known to play roles in inner ear development of higher vertebrates. In zebrafish, there are several reports showing that members of the BMP family are expressed in the otic vesicle. We have isolated a novel zebrafish mutant gallery, which affects the development of the semicircular canal. Gallery merely forms the lateral and the immature anterior protrusion, and does not form posterior and ventral protrusions. We found that the expression of bmp2b and bmp4, both expressed in the normal optic vesicle at the protrusion stage, are extremely upregulated in the otic vesicle of gallery. To elucidate the role of BMPs in the development of the inner ear of zebrafish, we have applied excess BMP to the wild‐type otic vesicle. The formation of protrusions was severely affected, and in some cases, they were completely lost in BMP4‐treated embryos. Furthermore, the protrusions in gallery treated with Noggin were partially rescued. These data indicate that BMP4 plays an important role in the development of protrusions to form semicircular canals.
Skeletogenesis by transfated secondary mesenchyme cells is dependent on extracellular matrix–ectoderm interactions in Paracentrotus lividus sea urchin embryos
Authors: Kiyomoto Masato , Zito Francesca , Costa Caterina , Poma Veronica , Sciarrino Serafina , Matranga Valeria
Published: August 2010

In the sea urchin embryo, primary mesenchyme cells (PMCs) are committed early in development to direct skeletogenesis, provided that a permissive signal is conveyed from adjacent ectoderm cells. We showed that inhibition of extracellular matrix (ECM)–ectoderm cells interaction, by monoclonal antibodies (mAb) to Pl‐nectin, causes an impairment of skeletogenesis and reduced expression of Pl‐SM30, a spicule‐specific matrix protein. When PMCs are experimentally removed, some secondary mesenchyme cells (SMCs) switch to skeletogenic fate. Here, for the first time we studied SMC transfating in PMC‐less embryos of Paracentrotus lividus. We observed the appearance of skeletogenic cells within 10 h of PMCs removal, as shown by binding of wheat germ agglutinin (WGA) to cell surface molecules unique to PMCs. Interestingly, the number of WGA‐positive cells, expressing also msp130, another PMC‐specific marker, doubled with respect to that of PMCs present in normal embryos, though the number of SM30‐expressing cells remained constant. In addition, we investigated the ability of SMCs to direct skeletogenesis in embryos exposed to mAbs to Pl‐nectin after removal of PMCs. We found that, although phenotypic SMC transfating occurred, spicule development, as well as Pl‐SM30‐expression was strongly inhibited. These results demonstrate that ectoderm inductive signals are necessary for transfated SMCs to express genes needed for skeletogenesis.
Expression, phosphorylation, and mRNA‐binding of heterogeneous nuclear ribonucleoprotein K in Xenopus oocytes, eggs, and early embryos
Authors: Iwasaki Tetsushi , Koretomo Yuta , Fukuda Teppei , Paronetto Maria Paola , Sette Claudio , Fukami Yasuo , Sato Ken‐ichi
Published: August 2010

Here we show that heterogeneous nuclear ribonucleoprotein K (hnRNP K), a member of the K homology domain‐containing proteins, is expressed in Xenopus immature oocytes, unfertilized eggs, and early embryos. Fertilization or egg activation treatment involving upregulation of the egg tyrosine kinase Src promotes a rapid and transient tyrosine phosphorylation of hnRNP K. HnRNP K is also phosphorylated on serine/threonine residues in unfertilized eggs, dephosphorylated after fertilization, and re‐phosphorylated during the premitotic phase of early embryogenesis. In vitro, Src and mitogen‐activated protein kinase (MAPK) were capable of phosphorylating hnRNP K on tyrosine and serine/threonine residues, respectively. In support of this, pretreatment of oocytes, eggs, or embryos with inhibitors for Src (PP2) and MAPK (U0126) blocked effectively the phosphorylation of hnRNP K. We also identify some maternal mRNAs that coimmunoprecipitate with hnRNP K in unfertilized eggs. Specific binding of these mRNAs to hnRNP K was verified by reverse transcriptase–polymerase chain reaction (RT–PCR). In addition, real‐time PCR analyses revealed a subset of the mRNAs whose binding to hnRNP K might be up or downregulated in activated eggs. In vitro binding assay with the use of poly U monopolymeric RNA‐coupled beads demonstrated that the RNA‐binding property of hnRNP K is negatively regulated by tyrosine phosphorylation and positively or neutrally regulated by serine/threonine phosphorylation. Taken together, it is attractive to suggest that hnRNP K is in association with certain pools of maternal mRNAs whose translational activation are modulated by the Src/MAPK phosphorylation of hnRNP K during oocyte‐egg‐embryo transition.
Isolation and characterization of Kit‐independent melanocyte precursors induced in the skin of Steel factor transgenic mice
Authors: Kawaguchi Atsushi , Chiba Kairi , Tanimura Yu , Motohashi Tsutomu , Aoki Hitomi , Takeda Tomoko , Hayashi Shin‐Ichi , Shimizu Katsuji , Kunisada Takahiro
Published: August 2010

Steel factor (SLF, also called KIT‐ligand, mast cell growth factor, or stem cell factor) acting through the tyrosine kinase receptor KIT is thought to be indispensable for the early phase of melanocyte development both in vivo and in vitro. In the present study, Kit‐independent precursor cells were generated in mice expressing exogenous SLF in their skin keratinocytes and were detected as pigmented spots after administration of Kit function‐blocking antibody. We successfully purified these precursor or stem cells as Kit+CD45− cells by flow cytometry. The purified cells showed normal but delayed differentiation into mature melanocytes, indicating the immature nature of Kit‐independent precursors. The Kit‐independent interfollicular population generated in SLF transgenic mice was suggested to be the counterpart of the follicular melanocyte stem cell based on the Kit‐independent nature for their survival.
Migration of mesenchymal cell fated to blastema is necessary for fish fin regeneration
Authors: Nakatani Yuki , Nishidate Masanobu , Fujita Misato , Kawakami Atsushi , Kudo Akira
Published: August 2010

Urodeles and fish have higher regeneration ability in a variety of tissues and organs than do other vertebrate species including mammals. Though many studies have aimed at identifying the cellular and molecular basis for regeneration, relatively little is known about the detailed cellular behaviors and involved molecular basis. In the present study, a small molecule inhibitor was used to analyzed the role of phosphoinositide 3‐kinase (PI3K) signaling during regeneration. We showed that the inhibitor disrupted the formation of blastema including the expression of characteristic genes. The failure of blastema formation was due to the impaired migration of mesenchymal cells to the distal prospective blastema region, although it had a little affect on cell cycle activation in mesenchymal cells. Moreover, we found that the epidermal remodeling including cell proliferation, distal cell migration and Akt phosphorylation was also affected by the inhibitor, implying a possible involvement of epidermis for proper formation of blastema. From these data, we propose a model in which distinct signals that direct the cell cycle activation, mesenchymal cell migration and epidermal remodeling coordinate together to accomplish the correct blastema formation and regeneration.
R‐spondin2 expression in the apical ectodermal ridge is essential for outgrowth and patterning in mouse limb development
Authors: Aoki Motoko , Kiyonari Hiroshi , Nakamura Harukazu , Okamoto Hitoshi
Published: August 2010

Mouse R‐spondin2 (Rspo2) is a member of the R‐spondin protein family, which is characterized by furin‐like cysteine‐rich domains and a thrombospondin type 1 repeat. R‐spondin is a secreted molecule that activates Wnt/β‐catenin signaling. Rspo2‐deficient mice were generated to investigate the function of mouse Rspo2 during embryonic development. The homozygous mutant forelimb showed defects in distal phalanges and nail structures, and the digits were anomalous in shape. The homozygous mutant hindlimb showed more severe malformations, including lack of digits and zeugopod components. Rspo2 is expressed in the apical ectodermal ridge (AER) of the developing limb. Fgf8 expression in the AER was significantly lower in the homozygous mutant forelimb than in the wild‐type forelimb and it was disturbed along the dorsoventral axis. In the homozygous mutant hindlimb, Fgf8 and Fgf4 expression in the posterior AER and Sonic hedgehog expression in the zone of polarizing activity (ZPA) were reduced. The homozygous mutant hindlimb also showed expansion of Wnt7a expression in the dorsal ectoderm toward the ventral side. This study shows that Rspo2 is critical for maintenance of the AER and for growth and patterning in limb development.
Signaling pathways in ascidian oocyte maturation: The role of cyclic AMP and follicle cells in germinal vesicle breakdown
Authors: Lambert Charles C.
Published: August 2010

Many ascidian oocytes undergo ‘spontaneous’ germinal vesicle breakdown (GVBD) when transferred from the ovary to normal pH 8.2 sea water (SW); however, low pH inhibits GVBD, which can then be stimulated while remaining in the low pH SW. Oocytes of Boltenia villosa blocked from GVBD by pH 4 SW undergo GVBD in response to permeant cyclic AMP (8‐bromo‐cyclic AMP), phosphodiesterase inhibitors (isobutylmethylxanthine and theophylline) or the adenylyl cyclase activator forskolin. This suggests that cAMP increases during GVBD. Removal of the follicle cells or addition of a protease inhibitor inhibits GVBD in response to raised pH but not to forskolin, theophylline or 8 bromo‐cAMP. Isolated follicle cells in low pH SW release protease activity in response to an increase in pH. These studies imply that the follicle cells release protease activity, which either itself stimulates an increase in oocyte cAMP level or reacts with other molecules to stimulate this process. Studies with the mitogen‐activated protein (MAP) kinase inhibitors U0126 and CI 1040 suggest that MAP kinase is not involved in GVBD. The Cdc25 inhibitor NSC 95397 inhibits GVBD at 200 nm in a reversible manner.
Analysis of the expression and function of Wnt‐5a and Wnt‐5b in developing and regenerating axolotl (Ambystoma mexicanum) limbs
Authors: Ghosh Sukla , Roy Stéphane , Séguin Carl , Bryant Susan V. , Gardiner David M.
Published: August 2010

Urodele amphibians are unique adult vertebrates because they are able to regenerate body parts after amputation. Studies of urodele limb regeneration, the key model system for vertebrate regeneration, have led to an understanding of the origin of blastema cells and the importance of positional interactions between blastema cells in the control of growth and pattern formation. Progress is now being made in the identification of the signaling pathways that regulate dedifferentiation, blastema morphogenesis, growth and pattern formation. Members of the Wnt family of secreted proteins are expressed in developing and regenerating limbs, and have the potential to control growth, pattern formation and differentiation. We have studied the expression of two non‐canonical Wnt genes, Wnt‐5a and Wnt‐5b. We report that they are expressed in equivalent patterns during limb development and limb regeneration in the axolotl (Ambystoma mexicanum), and during limb development in other tetrapods, implying conservation of function. Our analysis of the effects of ectopic Wnt‐5a expression is consistent with the hypothesis that canonical Wnt signaling functions during the early stages of regeneration to control the dedifferentiation of stump cells giving rise to the regeneration‐competent cells of the blastema.
Cadherins in neuronal morphogenesis and function
Authors: Suzuki Sachihiro C. , Takeichi Masatoshi
Published: August 2010

Classic cadherins represent a family of calcium‐dependent homophilic cell–cell adhesion molecules. They confer strong adhesiveness to animal cells when they are anchored to the actin cytoskeleton via their cytoplasmic binding partners, catenins. The cadherin/catenin adhesion system plays key roles in the morphogenesis and function of the vertebrate and invertebrate nervous systems. In early vertebrate development, cadherins are involved in multiple events of brain morphogenesis including the formation and maintenance of the neuroepithelium, neurite extension and migration of neuronal cells. In the invertebrate nervous system, classic cadherin‐mediated cell–cell interaction plays important roles in wiring among neurons. For synaptogenesis, the cadherin/catenin system not only stabilizes cell–cell contacts at excitatory synapses but also assembles synaptic molecules at synaptic sites. Furthermore, this system is involved in synaptic plasticity. Recent studies on the role of individual cadherin subtypes at synapses indicate that individual cadherin subtypes play their own unique role to regulate synaptic activities.
Determinative roles of FGF and Wnt signals in iris‐derived lens regeneration in newt eye
Authors: Hayashi Toshinori , Mizuno Nobuhiko , Kondoh Hisato
Published: August 2010

Total regeneration of experimentally excised lens from the dorsal part of the iris‐pigmented epithelium of newts has been a key model of tissue regeneration via cells originating from a foreign tissue. Due to the strict spatial restriction of the lens origin in the newt iris, it has often been assumed that only the dorsal iris cells are endowed with an intrinsic potential to give rise to lens tissues. However, our reinvestigation of the process revealed completely different mechanisms underlying lens regeneration and its spatial restriction, comprising the following two steps: (i) Fibroblast growth factor (FGF) 2‐dependent proliferation of iris‐pigmented epithelium and activation of early lens genes (Pax6, Sox2, MafB) over the entire circumference of the iris; and (ii) dorsal iris‐restricted activation of the canonical Wnt signals (involving Wnt2b and Frizzeld4) that leads to localized expression of late lens genes (Prox1, Sox1, β‐crystallin). Injection of FGF2 into normal eyes specifically elicited the second lens development from the dorsal iris, and the administration of recombinant Wnt3a to the cultured iris‐pigmented epithelium caused even ventral iris‐derived lens development. Thus, it is concluded that the regulation of FGF2 and Wnt signals is a determinative of the iris‐derived lens regeneration in the newt eye.
Shedding light on developmental gene regulation through the lens
Authors: Kondoh Hisato
Published: August 2010

My group has long studied transcriptional gene regulation involved in cell differentiation, employing lens cell differentiation as a model. In this article, our progress over the last quarter of a century in deciphering the principles involved in developmental gene regulation is described, outlining concurrent advancement in relevant branches of developmental biology.
Novel genes involved in canonical Wnt/β‐catenin signaling pathway in early Ciona intestinalis embryos
Authors: Wada Shuichi , Hamada Mayuko , Kobayashi Kenji , Satoh Nori
Published: August 2010

We report here characterization of five genes for novel components of the canonical Wnt/β‐catenin signaling pathway. These genes were identified in the ascidian Ciona intestinalis through a loss‐of‐function screening for genes required for embryogenesis with morpholinos, and four of them have counterparts in vertebrates. The five genes we studied are as follows: Ci‐PGAP1, a Ciona orthologue of human PGAP1, which encodes GPI (glycosylphosphatidylinositol) inositol‐deacylase, Ci‐ZF278, a gene encoding a C2H2 zinc‐finger protein, Ci‐C10orf11, a Ciona orthologue of human C10orf11 that encodes a protein with leucine‐rich repeats, Ci‐Spatial/C4orf17, a single counterpart for two human genes Spatial and C4orf17, and Ci‐FLJ10634, a Ciona orthologue of human FLJ10634 that encodes a member of the J‐protein family. Knockdown of each of the genes mimicked β‐catenin knockdown and resulted in suppression of the expression of β‐catenin downstream genes (Ci‐FoxD, Ci‐Lhx3, Ci‐Otx and Ci‐Fgf9/16/20) and subsequent endoderm formation. For every gene, defects in knockdown embryos were rescued by overexpression of a constitutively active form, but not wild‐type, of Ci‐β‐catenin. Dosage‐sensitive interactions were found between Ci‐β‐catenin and each of the genes. These results suggest that these five genes act upstream of or parallel to Ci‐β‐catenin in the Wnt/β‐catenin signaling pathway in early Ciona embryos.
Novel two‐step Ca2+ increase and its mechanisms and functions at fertilization in oocytes of the annelidan worm Pseudopotamilla occelata
Authors: Nakano Takeshi , Kyozuka Keiichiro , Deguchi Ryusaku
Published: August 2010

Mature oocytes of the annelidan worm Pseudopotamilla occelata have a wide perivitelline space between the oocyte surface and the vitelline envelope and are arrested at the first metaphase (MI). We found a novel two‐step Ca2+ increase in normally fertilized oocytes. The first Ca2+ increase originated at a cortex situated underneath a fertilizing sperm on the vitelline envelope, but failed to propagate beyond the center of the oocyte. The first localized Ca2+ increase was then followed by a larger Ca2+ increase starting from the whole oocyte cortex and spreading inwardly to the center. The first localized Ca2+ increase at fertilization was suppressed by the phospholipase C inhibitor U73122, and a similar Ca2+ change was induced by inositol 1,4,5‐trisphosphate (IP3). On the other hand, the second global Ca2+ increase in fertilized oocytes was blocked by removal of external Ca2+ or the voltage‐gated Ca2+ channel blocker D‐600, and a similar Ca2+ change could be mimicked by addition of excess K+ only when external Ca2+ was present. These results suggest that the first localized Ca2+ increase and the second global Ca2+ increase at fertilization are regulated by Ca2+ release from IP3‐sensitive stores and Ca2+ influx via voltage‐gated Ca2+ channels, respectively. Our data also demonstrated that the localized Ca2+ increase induces the formation of large cytoplasmic protrusion, which helps the fertilizing sperm to enter the oocyte, whereas the following global Ca2+ increase is a prerequisite for the retraction of the cytoplasmic protrusion and the resumption of meiosis from MI.
Wnt signaling in retinal stem cells and regeneration
Authors: Kubo Fumi , Nakagawa Shinichi
Published: August 2010

In the vertebrate retina, stem cells with prolonged proliferative capacities reside in the most peripheral region, the ciliary marginal zone (CMZ), and they persist even after the functional eye has formed. These stem cells contribute to the formation of the retinal structures during the postnatal period in vivo, or can expand as neurospheres in vitro. Despite the wealth of anatomical descriptions of the characteristics of CMZ cells, molecular mechanisms for their specification or maintenance have long been uncharacterized. Recent studies provide evidence that certain secreted signaling molecules act as key regulators at multiple steps during these processes. In this review, we discuss the molecular basis for the regulation of retinal stem cells and their related cell types, especially focusing on the role of Wnt signaling.