bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of systematic review methods to qualitative research: practical issues
Authors: Jones Myfanwy Lloyd
Published: October 2004

Aim.  This paper explores practical methodological issues which arise from the application of systematic review and meta‐synthesis techniques to qualitative research studies in the context of a pragmatic health services research question.

Background.  The emphasis on, and volume of, qualitative research is increasing. As a result, there is a need to integrate and disseminate qualitative research findings. However, relatively little has been written about the methodology of systematically reviewing and meta‐synthesizing qualitative research studies, and about the practical issues which arise in the course of these processes.

Methods.  A systematic review and meta‐synthesis was undertaken of qualitative research studies reporting data relevant to the pragmatic health services research question: ‘What factors facilitate or impede role development and/or effective practice as a clinical nurse specialist, nurse practitioner, advanced nurse practitioner or consultant nurse based in acute hospital settings?’

Findings.  The identification of relevant studies is substantially more time‐consuming than the identification of relevant studies for a systematic review of randomized controlled trials. A substantially larger proportion of papers has to be retrieved for full reading. Articles with unclear titles which lack abstracts cannot be dismissed as irrelevant. Study appraisal and data analysis, being iterative processes, are also more time‐consuming than the appraisal and meta‐analysis of quantitative studies. It may be possible to reduce the frustrations inherent in the distance between the reviewer and the participants in the primary research by using full project reports rather than published articles.

Conclusions.  Conducting a systematic review and meta‐synthesis of qualitative research studies is a rewarding but demanding activity, and adequate time and resources must be made available. Some recommendations are made which may facilitate those processes.
Assessing mothers’ concerns in the postpartum period: methodological issues
Authors: Lugina Helen I. , Nyström Lennarth , Christensson Kyllike , Lindmark Gunilla
Published: October 2004

Aim.  This paper reports a study evaluating the sensitivity of a semi‐structured interview schedule and card sort methods in assessing postpartum concerns of women.

Background.  Several methods have been used to assess postpartum maternal concerns and the process of becoming a mother, but few studies have evaluated the methods with respect to their sensitivity for obtaining information.

Method.  A cohort of mothers was followed‐up at one (n = 110) and 6 weeks (n = 83) after childbirth in Dar es Salaam, Tanzania. Women with a minimum of 7 years of primary education were interviewed and they also sorted cards. Those with less fewer than 7 years of primary education were interviewed only. The methods were used in alternate order to assess method interaction.

Results.  In the interviews at 1 week, mothers more often expressed worry and interest related to the baby or themselves when they had sorted cards first. The extent to which women expressed worry and interest about specific baby‐ and mother‐related topics was generally higher for women who had sorted cards before the interview at both 1 and 6 weeks. Independent of whether they were interviewed only, interviewed after sorting cards or before, mothers more often expressed a higher degree of interest than of worry about the baby and self at both 1 and 6 weeks. The order of the data collection methods did not influence the way women sorted cards as being worries and interests.

Conclusion.  Compared to interview using a semi‐structured interview schedule, our findings suggest that the card sort is more sensitive in obtaining information about women's concerns. Although the interview method has the advantage of reaching less educated people, the card sort is a technique that is associated with fewer barriers and is a more participatory method for those who can use it.
Nursing students’ beliefs about poverty and health
Authors: Reutter Linda I. , Sword Wendy , Meagher‐Stewart Donna , Rideout Elizabeth
Published: October 2004

Purpose.  This paper examines baccalaureate nursing students’ beliefs about the relationship between poverty and health, and the factors that influence these beliefs.

Background.  The relationship between poverty and health is well established, and poverty remains a persistent problem in many industrialized nations. Nurses’ understanding of how poverty influences health will affect how they interact with individual clients as well as the strategies they employ to address poverty‐related issues. No studies have examined nursing students’ understandings of how poverty influences health and the factors that influence that understanding.

Methods.  A cross‐sectional survey of a random sample (n = 740) of basic baccalaureate nursing students was conducted in three Canadian universities in 2000. Students completed a 59‐item questionnaire eliciting data on demographic variables, personal and educational exposure to poverty, beliefs about the relationship between poverty and health (myth, drift, behavioural, structural), and attitudes to poverty.

Results.  Students were most likely to adhere to a structural explanation of the relationship between poverty and health. Very little of the variance in myth and drift explanations was accounted for by course or personal exposure, programme level, age, and attitudes toward poverty. Greater course exposure and more positive attitudes toward the poor predicted support for the structural explanation. Support for the behavioural explanation was influenced by attitudes toward the poor and, to a lesser extent, by course exposure, age, and programme level.

Conclusion.  Students would benefit from greater exposure to poverty through coursework that emphasizes the structural factors contributing to poverty and its negative health consequences. Classroom experience should be complemented with clinical placements that provide students with opportunities to interact with families living in poverty and to work collaboratively with others to address the causes and consequences of poverty at community and policy levels.
Manipulation of DET1 expression in tomato results in photomorphogenic phenotypes caused by post‐transcriptional gene silencing
Authors: Davuluri Ganga Rao , van Tuinen Ageeth , Mustilli Anna Chiara , Manfredonia Alessandro , Newman Robert , Burgess Diane , Brummell David A. , King Stephen R. , Palys Joe , Uhlig John , Pennings Henk M. J. , Bowler Chris
Published: October 2004

The tomato HIGH PIGMENT‐2 gene encodes an orthologue of the Arabidopsis nuclear protein DE‐ETIOLATED 1 (DET1). From genetic analyses it has been proposed that DET1 is a negative regulator of light signal transduction, and recent results indicate that it may control light‐regulated gene expression at the level of chromatin remodelling. To gain further understanding about the function of DET1 during plant development, we generated a range of overexpression constructs and introduced them into tomato. Unexpectedly, we only observed phenotypes characteristic of DET1 inactivation, i.e. hyper‐responsiveness to light. Molecular analysis indicated in all cases that these phenotypes were a result of suppression of endogenous DET1 expression, due to post‐transcriptional gene silencing. DET1 silencing was often lethal when it occurred at relatively early stages of plant development, whereas light hyper‐responsive phenotypes were obtained when silencing occurred later on. The appearance of phenotypes correlated with the generation of siRNAs but not DNA hypermethylation, and was most efficient when using constructs with mutations in the DET1 coding sequence or with constructs containing only the 3′‐terminal portion of the gene. These results indicate an important function for DET1 throughout plant development and demonstrate that silencing of DET1 in fruits results in increased carotenoids, which may have biotechnological potential.
Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation
Authors: Walter Michael , Chaban Christina , Schütze Katia , Batistic Oliver , Weckermann Katrin , Näke Christian , Blazevic Dragica , Grefen Christopher , Schumacher Karin , Oecking Claudia , Harter Klaus , Kudla Jörg
Published: October 2004

Dynamic networks of protein–protein interactions regulate numerous cellular processes and determine the ability to respond appropriately to environmental stimuli. However, the investigation of protein complex formation in living plant cells by methods such as fluorescence resonance energy transfer has remained experimentally difficult, time consuming and requires sophisticated technical equipment. Here, we report the implementation of a bimolecular fluorescence complementation (BiFC) technique for visualization of protein–protein interactions in plant cells. This approach relies on the formation of a fluorescent complex by two non‐fluorescent fragments of the yellow fluorescent protein brought together by association of interacting proteins fused to these fragments (Hu et al., 2002). To enable BiFC analyses in plant cells, we generated different complementary sets of expression vectors, which enable protein interaction studies in transiently or stably transformed cells. These vectors were used to investigate and visualize homodimerization of the basic leucine zipper (bZIP) transcription factor bZIP63 and the zinc finger protein lesion simulating disease 1 (LSD1) from Arabidopsis as well as the dimer formation of the tobacco 14‐3‐3 protein T14‐3c. The interaction analyses of these model proteins established the feasibility of BiFC analyses for efficient visualization of structurally distinct proteins in different cellular compartments. Our investigations revealed a remarkable signal fluorescence intensity of interacting protein complexes as well as a high reproducibility and technical simplicity of the method in different plant systems. Consequently, the BiFC approach should significantly facilitate the visualization of the subcellular sites of protein interactions under conditions that closely reflect the normal physiological environment.
Molecular dissection of plant cytokinesis and phragmoplast structure: a survey of GFP‐tagged proteins
Authors: Damme Daniël Van , Bouget François‐Yves , Poucke Kris Van , Inzé Dirk , Geelen Danny
Published: October 2004

To identify molecular players implicated in cytokinesis and division plane determination, the Arabidopsis thaliana genome was explored for potential cytokinesis genes. More than 100 open reading frames were selected based on similarity to yeast and animal cytokinesis genes, cytoskeleton and polarity genes, and Nicotiana tabacum genes showing cell cycle‐controlled expression. The subcellular localization of these proteins was determined by means of GFP tagging in tobacco Bright Yellow‐2 cells and Arabidopsis plants. Detailed confocal microscopy identified 15 proteins targeted to distinct regions of the phragmoplast and the cell plate. EB1‐ and MAP65‐like proteins were associated with the plus‐end, the minus‐end, or along the entire length of microtubules. The actin‐binding protein myosin, the kinase Aurora, and a novel cell cycle protein designated T22, accumulated preferentially at the midline. EB1 and Aurora, in addition to other regulatory proteins (homologs of Mob1, Sid1, and Sid2), were targeted to the nucleus, suggesting that this organelle operates as a coordinating hub for cytokinesis.
Granulocyte adsorptive apheresis for leg ulcers complicated by rheumatoid arthritis: a report on three successfully treated cases
Authors: Mori Shinya , Nagashima Masakazu , Yoshida Kazuhiro , Yoshino Kouji , Aoki Mikako , Kawana Seiji , Hirata Ichiro , Saniabadi Abby , Yoshino Shinichi
Published: October 2004

Background  The treatment of inflammatory leg ulcers complicated by rheumatoid arthritis (RA), which are unresponsive to conventional care, can be frustrating. Furthermore, as granulocytes and monocytes (GM) are major sources of inflammatory cytokines, they have the potential to initiate and perpetuate inflammatory skin lesions. Accordingly, a recent study reported the remission of pyoderma gangrenosum following the reduction of activated peripheral blood GM by adsorptive apheresis (GMA).

Methods  In this clinical study, we applied GMA to three cases, each with one leg ulcer below the knee and RA. The ulcers had not responded to conventional therapy, including disinfection, dressing, and antimicrobials, and therefore were thought to represent inflammatory vasculitic lesions. GMA was performed using a column with a capacity of 335 mL, filled with cellulose acetate beads that selectively adsorb granulocytes and monocytes/macrophages (Adacolumn). Each patient received one GMA session/week for five consecutive weeks. The duration of one session was 60 min, with a flow rate of 30 mL/min.

Results  The ulcers began to recede after two GMA sessions and, by the end of the fifth session, the ulcers in all three patients had healed. No recurrence has been observed up to the time of this report. The treatment was well tolerated and no severe side‐effects were observed.

Conclusions  GMA, which depletes activated neutrophils and monocytes/macrophages, appears to be effective for inflammatory skin ulcers which do not respond to conventional medications.
Insect bite‐like reaction associated with mantle cell lymphoma: a report of two cases and review of the literature
Authors: Dodiuk‐Gad Roni P. , Dann Eldad J. , Bergman Reuven
Published: October 2004

Background  An insect bite‐like reaction and exaggerated reactions to insect bites are nonspecific phenomena described primarily in association with chronic lymphocytic leukemia (CLL), but also with other hematological malignancies. Two cases of mantle cell lymphoma (MCL), one associated with an insect bite‐like reaction and the other with a true hypersensitivity to mosquito bites, have previously been reported in the English language literature. The pathogenesis of the skin eruption may be related to the release of different cytokines that also trigger an IgE elevation and dermal eosinophils.

Case report  We describe two additional cases of MCL associated with an insect bite‐like reaction. One patient had been diagnosed with MCL 4.5 years prior to the appearance of the skin eruption, and in the other patient the skin symptoms preceded the diagnosis of the MCL by 2 years and led to its diagnosis.

Conclusions  Insect bite‐like reaction may appear in patients with MCL. It is important to recognize this entity because it may be the presenting sign of MCL.
Immunohistochemical study of serotonin in lesions of chronic eczema
Authors: Huang Jianguo , Li Guiming , Xiang Jihu , Yin Daochun , Chi Ridong
Published: October 2004

Background  Inflammatory mediators participate in the pathogenesis and pathophysiology of skin disease (e.g. eczema). Although some investigators have suggested that serotonin may participate in the eczematous process, there has been little substantial evidence. Our objective was to study further the participation of serotonin in the pathogenesis of chronic eczema.

Methods  Immunohistochemical techniques were used to study the expression of serotonin in the lesions of patients with chronic eczema.

Results  There was a significantly stronger expression of serotonin in the prickle cells, sweat gland cells, sebaceous gland cells, and hair roots of the lesions in patients with chronic eczema. There was no positive expression of serotonin in normal skin.

Conclusions  Serotonin is probably important in the pathogenesis of chronic eczema. We demonstrated alterations in the expression of serotonin in chronic eczema lesions, which may in turn provide new insight into the pathogenesis of chronic eczema. Serotonin, along with cytokines, T lymphocytes, B lymphocytes, and macrophages, participates in the immune response and influences the inflammatory process by means of keratinocytes.
Sweet's syndrome revealing relapsing polychondritis
Authors: Astudillo L. , Launay F. , Lamant L. , Sailler L. , Bazex J. , Couret B. , Arlet‐Suau E.
Published: October 2004

Sweet's syndrome is extremely rare in relapsing polychondritis. We report a rare case of Sweet's syndrome revealing relapsing polychondritis. A 77‐year‐old man presented with fever, associated with respiratory symptoms and non pruritic, painful, erythematous, papules and plaques mainly on the arms and legs. Skin biopsy of the arm found a neutrophilic dermal infiltrate consistent with Sweet's syndrome. A month and a half after admission, the patient presented with ear chondritis and dysphonia. Biopsy of the ear cartilage found a significant perichondral inflammatory cells infiltrate. The diagnosis of relapsing polychondritis was established and a steroid treatment was instituted with a complete regression of the cutaneous signs and chondritis.
Primary malignant melanoma of the oral cavity: a case report
Authors: López Ortega Karem , Soares de Araújo Ney , Bitu de Souza Fabricio , Magalhães Marina H. C. G.
Published: October 2004

A 43‐year‐old white man was referred to the Special Care Dentistry Center of the School of Dentistry, University of São Paulo, Brazil, for the diagnosis of an extensive nodular lesion of the maxillary gingiva.

The patient complained that his left maxilla had swollen over the last 4 months, with some exudation from the gingival crevice, sporadic bleeding, and slight tooth mobility, but no pain.

An extra‐oral examination confirmed expressive swelling of the left side of the face (Fig. 1A). Palpation disclosed bilateral enlargement of the submandibular lymph nodes.

Clinical and radiographic characteristics of the oral malignant melanoma. (A) Swelling of the left side of the face. (B) Extensive nodular mass on the maxillary gingiva. (C) Palatal view of the tumor with a focal pigmented area. (D) Computed tomography scan showing extensive involvement of the anterior maxilla and pterygoid muscle

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An intra‐oral examination showed an extensive, reddish, nodular mass (around 11 cm in diameter) that extended from the last left maxillary molar to the right maxillary incisor, covered by a mucosa that was ulcerated in some areas (Fig. 1B,C). Palpation revealed a painless, soft, bleeding tissue that seemed to arise in the periodontal ligaments, extending to the palate and vestibular area. A small pigmented spot was found in the palatal mass.

Histopathologically, the biopsy revealed a proliferation of neoplastic cells that exhibited a wide variety of shapes, including spindle, plasmacytoid, and epithelioid forms. The atypical cells showed enlarged and pleomorphic nuclei. Mitotic activity and pigmented areas were observed (Fig. 2A,B). Immunohistochemistry was used to establish the final diagnosis. The tumor cells strongly expressed S100 protein, gp100 (HMB‐45), melan A, and tyrosine antibodies (Fig. 2C,D).

Microscopic biopsy findings of the oral malignant melanoma. (A) Low‐power photomicrograph of the mucosa reveals the proliferation of abnormal melanocytes in the connective tissue (hematoxylin and eosin, ×20). (B) Cells of fairly uniform size and shape, arranged in an alveolar pattern (hematoxylin and eosin, ×40). (C) S‐100 protein is expressed by all tumor cells (×40). (D) Melan A protein is expressed more frequently in the pigmented areas (×40)

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With the diagnosis of malignant melanoma, the patient was referred to an oncologist for treatment. As computed tomography revealed that the lesion was deeply inserted into the skull surface (Fig. 1D), surgical intervention was not possible. The patient underwent radiotherapy, but died 14 months later.
Formable acrylic treatment for ingrowing nail with gutter splint and sculptured nail
Authors: Arai Hiroko , Arai Takeo , Nakajima Hiroshi , Haneke Eckart
Published: October 2004

Background  The treatment of choice for an ingrowing nail has been surgical rather than nonsurgical. Yet, surgical treatments are far from successful, cause pain and patient apprehension, and leave disfigurement. Further, there is misunderstanding about the disease pathophysiology.

Objective  To demonstrate the benefits of a noninvasive method of treatment for an ingrowing nail using gutter splint and formable acrylics and to present a current understanding of the disease pathophysiology.

Methods  From a total of 541 cases of ingrowing nails treated, full follow‐up data were obtained between January 1979 and March 2002. Formable acrylic treatments were carried out in 106 cases treated with acrylic‐affixed gutter splint, 17 cases with sculptured nails, and 28 cases in which the two treatments were combined. These were then compared with 233 cases treated with adhesive tape‐attached gutter splint and the remainder with other conservative modalities.

Results  Acrylic treatment with gutter splint and sculptured nail was found to be vastly superior to the other methods described, especially in the ability to firmly affix the gutter splint and sculptured nail for the extended period required for treatment. The treatment leads to a complete remission with almost instant alleviation of pain, with no disfigurement, while allowing for the resumption of daily activities.

Conclusion  Conservative methods utilizing formable acrylics are highly beneficial in the treatment of an ingrowing nail and should be viewed as the first treatment option.
A kinetic study of sugarcane sucrose synthase
Authors: Schäfer Wolfgang E. , Rohwer Johann M. , Botha Frederik C.
Published: October 2004

The kinetic data on sugarcane (Saccharum spp. hybrids) sucrose synthase (SuSy, UDP‐glucose: d‐fructose 2‐α‐d‐glucosyltransferase, EC 2.4.1.13) are limited. We characterized kinetically a SuSy activity partially purified from sugarcane variety N19 leaf roll tissue. Primary plot analysis and product inhibition studies showed that a compulsory order ternary complex mechanism is followed, with UDP binding first and UDP‐glucose dissociating last from the enzyme. Product inhibition studies showed that UDP‐glucose is a competitive inhibitor with respect to UDP and a mixed inhibitor with respect to sucrose. Fructose is a mixed inhibitor with regard to both sucrose and UDP. Kinetic constants are as follows: Km values (mm, ± SE) were, for sucrose, 35.9 ± 2.3; for UDP, 0.00191 ± 0.00019; for UDP‐glucose, 0.234 ± 0.025 and for fructose, 6.49 ± 0.61. values were, for sucrose, 227 mm; for UDP, 0.086 mm; for UDP‐glucose, 0.104; and for fructose, 2.23 mm. Replacing estimated kinetic parameters of SuSy in a kinetic model of sucrose accumulation with experimentally determined parameters of the partially purified isoform had significant effects on model outputs, with a 41% increase in sucrose concentration and 7.5‐fold reduction in fructose the most notable. Of the metabolites included in the model, fructose concentration was most affected by changes in SuSy activity: doubling and halving of SuSy activity reduced and increased the steady‐state fructose concentration by about 42 and 140%, respectively. It is concluded that different isoforms of SuSy could have significant differential effects on metabolite concentrations in vivo, therefore impacting on metabolic regulation.
Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation
Authors: Albrecht Tanja , Haebel Sophie , Koch Anke , Krause Ulrike , Eckermann Nora , Steup Martin
Published: October 2004

Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a conserved tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix‐assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C‐terminal (His6) tag, was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide (residues 219–246 of the complete Glg2p sequence) was isolated that contained 4–25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography, mono‐ and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p‐producing cells accumulated approximately 30% less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogen‐related enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the bacterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, the eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis.
Cytokinin oxidase/dehydrogenase genes in barley and wheat
Authors: Galuszka Petr , Frébortová Jitka , Werner Tomáš , Yamada Mamoru , Strnad Miroslav , Schmülling Thomas , Frébort Ivo
Published: October 2004

The cloning of two novel genes that encode cytokinin oxidase/dehydrogenase (CKX) in barley is described in this work. Transformation of both genes into Arabidopsis and tobacco showed that at least one of the genes codes for a functional enzyme, as its expression caused a cytokinin‐deficient phenotype in the heterologous host plants. Additional cloning of two gene fragments, and an in silico search in the public expressed sequence tag clone databases, revealed the presence of at least 13 more members of the CKX gene family in barley and wheat. The expression of three selected barley genes was analyzed by RT‐PCR and found to be organ‐specific with peak expression in mature kernels. One barley CKX (HvCKX2) was characterized in detail after heterologous expression in tobacco. Interestingly, this enzyme shows a pH optimum at 4.5 and a preference for cytokinin ribosides as substrates, which may indicate its vacuolar targeting. Different substrate specificities, and the pH profiles of cytokinin‐degrading enzymes extracted from different barley tissues, are also presented.
Expression of the Drosophila melanogaster ATP synthase α subunit gene is regulated by a transcriptional element containing GAF and Adf‐1 binding sites
Authors: Talamillo Ana , Fernández‐Moreno Miguel Angel , Martínez‐Azorín Francisco , Bornstein Belén , Ochoa Pilar , Garesse Rafael
Published: October 2004

Mitochondrial biogenesis is a complex and highly regulated process that requires the controlled expression of hundreds of genes encoded in two separated genomes, namely the nuclear and mitochondrial genomes. To identify regulatory proteins involved in the transcriptional control of key nuclear‐encoded mitochondrial genes, we have performed a detailed analysis of the promoter region of the α subunit of the Drosophila melanogaster F1F0 ATP synthase complex. Using transient transfection assays, we have identified a 56 bp cis‐acting proximal regulatory region that contains binding sites for the GAGA factor and the alcohol dehydrogenase distal factor 1. In vitro mutagenesis revealed that both sites are functional, and phylogenetic footprinting showed that they are conserved in other Drosophila species and in Anopheles gambiae. The 56 bp region has regulatory enhancer properties and strongly activates heterologous promoters in an orientation‐independent manner. In addition, Northern blot and RT‐PCR analysis identified two α‐F1‐ATPase mRNAs that differ in the length of the 3′ untranslated region due to the selection of alternative polyadenylation sites.
Characterization of a cathepsin L‐associated protein in Artemia and its relationship to the FAS‐I family of cell adhesion proteins
Authors: Warner Alden H. , Pullumbi Ervin , Amons Reinout , Liu Liqian
Published: October 2004

We reported previously that the major cysteine protease in embryos and larvae of the brine shrimp, Artemia franciscana, is a heterodimeric protein consisting of a catalytic subunit (28.5 kDa) with a high degree of homology with cathepsin L, and a noncatalytic subunit (31.5 kDa) of unknown function. In the study reported here the noncatalytic subunit, or cathepsin L‐associated protein (CLAP), was separated from cathepsin L by chromatography on Mono S and found to contain multiple isoforms with pIs ranging from 5.9 to 6.1. Heterodimeric and monomeric cathepsin L showed similar activity between pH 5 and 6.5, while the heterodimer was about twice as active as monomeric cathepsin L below pH 5. The heterodimer was more stable than the monomer between pH 6 and 7.4 and at 30–50 °C. Artemia CLAP and cathepsin L are present in nearly equimolar amounts at all stages in the life cycle and most abundant in encysted eggs and embyros. Moreover, CLAP, either free or as a complex with cathepsin L, was resistant to hydrolysis by cathepsin L. Two clones coding for CLAP were isolated from an Artemia embryo cDNA library and sequenced. Both clones have nearly identical open reading frames, but show differences at the 5′‐ and 3′‐termini. Each cDNA clone has an extensive 3′‐untranslated region containing 70–72% A+T. The deduced amino acid sequence of CLAP cDNA revealed two domains which were very similar to domains in fasciclin I and other cell adhesion proteins. The nucleotide sequences of clones 1 and 2 have been entered into the NCBI database (AY307377 and AY462276). This study supports the view that the noncatalytic subunit of the heterodimeric cysteine protease in Artemia stabilizes cathepsin L at various pH and temperatures normally inconsistent with cathepsin L from other organisms, and that CLAP serves as a docking mechanism for cathepsin L at nonlysosomal sites in Artemia embryos.
Investigation of the contribution of histidine 119 to the conduction of protons through human Nox2
Authors: Mankelow Tosti J. , Hu X. Wen , Adams Kate , Henderson Lydia M.
Published: October 2004

The conduction of protons through human Nox2 has previously been shown to be dependent upon His115. Alignment of sequences for both animal and plant Nox proteins indicated that histidines 115 and 119 are both highly conserved, while His111 was conserved among animal homologues of Nox1–4. To investigate the possible role that these histidine residues might play in the conduction of protons through Nox2, we have introduced both paired and single mutations into these histidine residues. Each construct was used to generate a CHO cell line in which the expression of the mutated Nox2 was assessed. Nox2 was expressed in each of the CHO cell lines generated, however, the level of expression of H111/115L in CHO cells was lower and that of H111L very much reduced, compared to that of wild‐type Nox2. The arachidonic acid activated proton flux was absent in the CHO cell lines expressing the mutations of H111/115L, H111/119L or H115/119L, compared to that observed for wild‐type Nox2. Similarly only a small efflux of protons was observed from CHO cells expressing either H119L or H111L. In all cases the expected proton flux was elicited through the addition of the protonophore, carbonyl cyanide m‐chlorophenylhydrazone. Conclusions regarding the role of His111 in the conduction of protons cannot be drawn due to the reduced expression. We can, however, conclude that His119, in addition to His115, is required for the conduction of protons through Nox2. His119 has been identified as a highly conserved residue for which no function has previously been proposed.
Factors affecting habituation of PC12 cells to ATP
Authors: Keath J. Russel , Westhead Edward W.
Published: October 2004

Extracellular ATP triggers catecholamine secretion from PC12 cells by activating ionotropic purine receptors. Repeated stimulation by ATP leads to habituation of the secretory response. In this paper, we use amperometric detection to monitor the habituation of PC12 cells to multiple stimulations of ATP or its agonist. Cells habituate to 30 µm ATP slower than they do to 300 or 600 µm ATP. Modifying external Mg2+ affects the response of cells to 30 µm ATP, but does not affect habituation, suggesting that habituation does not necessarily correspond to either stimulus intensity or cellular response. Mg2+ affects the initial response of PC12 cells to 2MeSATP in a manner similar to ATP. Increasing external [Mg2+] to 3.0 mm, however, eliminates habituation to 2MeSATP. This habituation can be partially restored by costimulation with 100 µm UTP. Background application of UTP increases habituation to both ATP and 2MeSATP. This suggests that ATP‐sensitive metabotropic (P2Y) receptors play a role in the habituation process. Finally, although Ca2+ influx through voltage‐operated calcium channels does not appear to contribute to secretion during ATP stimulation, blocking these channels with nicardipine increases habituation. This suggests a role for voltage‐operated calcium channels in the habituation process.
Differential effects of RU486 reveal distinct mechanisms for glucocorticoid repression of prostaglandin E2 release
Authors: Chivers Joanna E. , Cambridge Lisa M. , Catley Matthew C. , Mak Judith C. , Donnelly Louise E. , Barnes Peter J. , Newton Robert
Published: October 2004

In A549 pulmonary cells, the dexamethasone‐ and budesonide‐dependent repression of interleukin‐1β‐induced prostaglandin E2 release was mimicked by the steroid antagonist, RU486. Conversely, whereas dexamethasone and budesonide were highly effective inhibitors of interleukin‐1β‐induced cyclooxygenase (COX)/prostaglandin E synthase (PGES) activity and COX‐2 expression, RU486 (< 1 µm) was a poor inhibitor, but was able to efficiently antagonize the effects of dexamethasone and budesonide. In addition, both dexamethasone and RU486 repressed [3H]arachidonate release, which is consistent with an effect at the level of phospholipase A2 activity. By contrast, glucocorticoid response element‐dependent transcription was unaffected by RU486 but induced by dexamethasone and budesonide, whilst dexamethasone‐ and budesonide‐dependent repression of nuclear factor‐κB‐dependent transcription was maximally 30–40% and RU486 (< 1 µm) was without significant effect. Thus, two pharmacologically distinct mechanisms of glucocorticoid‐dependent repression of prostaglandin E2 release are revealed. First, glucocorticoid‐dependent repression of arachidonic acid is mimicked by RU486 and, second, repression of COX/PGES is antagonized by RU486. Finally, whilst all compounds induced glucocorticoid receptor translocation, no role for glucocorticoid response element‐dependent transcription is supported in these inhibitory processes and only a limited role for glucocorticoid‐dependent inhibition of nuclear factor‐κB in the repression of COX‐2 is indicated.
The Y42H mutation in medium‐chain acyl‐CoA dehydrogenase, which is prevalent in babies identified by MS/MS‐based newborn screening, is temperature sensitive
Authors: O'Reilly Linda , Bross Peter , Corydon Thomas J. , Olpin Simon E. , Hansen Jakob , Kenney John M. , McCandless Shawn E. , Frazier Dianne M. , Winter Vibeke , Gregersen Niels , Engel Paul C , Storstein Andresen Brage
Published: October 2004

Medium‐chain acyl‐CoA dehydrogenase (MCAD) is a homotetrameric flavoprotein which catalyses the initial step of the β‐oxidation of medium‐chain fatty acids. Mutations in MCAD may cause disease in humans. A Y42H mutation is frequently found in babies identified by newborn screening with MS/MS, yet there are no reports of patients presenting clinically with this mutation. As a basis for judging its potential consequences we have examined the protein phenotype of the Y42H mutation and the common disease‐associated K304E mutation. Our studies of the intracellular biogenesis of the variant proteins at different temperatures in isolated mitochondria after in vitro translation, together with studies of cultured patient cells, indicated that steady‐state levels of the Y42H variant in comparison to wild‐type were decreased at higher temperature though to a lesser extent than for the K304E variant. To distinguish between effects of temperature on folding/assembly and the stability of the native enzyme, the thermal stability of the variant proteins was studied after expression and purification by dye affinity chromatography. This showed that, compared with the wild‐type enzyme, the thermostability of the Y42H variant was decreased, but not to the same degree as that of the K304E variant. Substrate binding, interaction with the natural electron acceptor, and the binding of the prosthetic group, FAD, were only slightly affected by the Y42H mutation. Our study suggests that Y42H is a temperature sensitive mutation, which is mild at low temperatures, but may have deleterious effects at increased temperatures.
Cloning, expression and interaction of human T‐cell receptors with the bacterial superantigen SSA
Authors: De Marzi Mauricio C. , Fernández Marisa M. , Sundberg Eric J. , Molinero Luciana , Zwirner Norberto W. , Llera Andrea S. , Mariuzza Roy A. , Malchiodi Emilio L.
Published: October 2004

Superantigens (SAgs) are a class of disease‐causing and immunostimulatory proteins of bacterial or viral origin that activate a large number of T‐cells through interaction with the Vβ domain of T‐cell receptors (TCRs). In this study, recombinant TCR β chains were constructed with human variable domains Vβ5.2, Vβ1 and Vβ2.1, expressed as inclusion bodies, refolded and purified. The Streptococcus pyogenes SAg SSA‐1 was cloned and expressed as a soluble periplasmic protein. SSA‐1 was obtained both as a monomer and a dimer that has an intermolecular disulfide bond. We analyzed the biological activity of the recombinant SAgs by proliferation assays. The results suggest that SSA dimerization occludes the TCR interaction site. Naturally occurring SSA dimerization was also observed in supernatants of S. pyogenes isolates. An SSA mutant [SSA(C26S)] was produced to eliminate the Cys responsible for dimerization. Affinity assays using a resonant biosensor showed that both the mutant and monomeric wild type SSA have affinity for human Vβ5.2 and Vβ1 with Kd of 9–11 µm with a fast kass and a moderately fast kdiss. In spite of the reported stimulation of Vβ2.1 bearing T‐cells by SSA, we observed no measurable interaction.
X‐ray structure of peptidyl‐prolyl cis–trans isomerase A from Mycobacterium tuberculosis
Authors: Henriksson Lena M. , Johansson Patrik , Unge Torsten , Mowbray Sherry L.
Published: October 2004

Peptidyl‐prolyl cis–trans isomerases (EC 5.2.1.8) catalyse the interconversion of cis and trans peptide bonds and are therefore considered to be important for protein folding. They are also thought to participate in processes such as signalling, cell surface recognition, chaperoning and heat‐shock response. Here we report the soluble expression of recombinant Mycobacterium tuberculosis peptidyl‐prolyl cis–trans isomerase PpiA in Escherichia coli, together with an investigation of its structure and biochemical properties. The protein was shown to be active in a spectrophotometric assay, with an estimated kcat/Km of 2.0 × 106 m−1·s−1. The X‐ray structure of PpiA was solved by molecular replacement, and refined to a resolution of 2.6 Å with R and Rfree values of 21.3% and 22.9%, respectively. Comparisons to known structures show that the PpiA represents a slight variation on the peptidyl‐prolyl cis–trans isomerase fold, previously not represented in the Protein Data Bank. Inspection of the active site suggests that specificity for substrates and cyclosporin A will be similar to that found for most other enzymes of this structural family. Comparison to the sequence of the second M. tuberculosis enzyme, PpiB, suggests that binding of peptide substrates as well as cyclosporin A may differ in that case.
Crystal structures of the human SUMO‐2 protein at 1.6 Å and 1.2 Å resolution
Authors: Huang Wen‐Chen , Ko Tzu‐Ping , Li Steven S.‐L , Wang Andrew H.‐J.
Published: October 2004

The SUMO proteins are a class of small ubiquitin‐like modifiers. SUMO is attached to a specific lysine side chain on the target protein via an isopeptide bond with its C‐terminal glycine. There are at least four SUMO proteins in humans, which are involved in protein trafficking and targeting. A truncated human SUMO‐2 protein that contains residues 9–93 was expressed in Escherichia coli and crystallized in two different unit cells, with dimensions of a = b = 75.25 Å, c = 29.17 Å and a = b = 74.96 Å, c = 33.23 Å, both belonging to the rhombohedral space group R3. They diffracted X‐rays to 1.6 Å and 1.2 Å resolution, respectively. The structures were determined by molecular replacement using the yeast SMT3 protein as a search model. Subsequent refinements yielded R/Rfree values of 0.169/0.190 and 0.119/0.185, at 1.6 Å and 1.2 Å, respectively. The peptide folding of SUMO‐2 consists of a half‐open β‐barrel and two flanking α‐helices with secondary structural elements arranged as ββαββαβ in the sequence, identical to those of ubiquitin, SMT3 and SUMO‐1. Comparison of SUMO‐2 with SUMO‐1 showed a surface region near the C terminus with significantly different charge distributions. This may explain their distinct intracellular locations. In addition, crystal‐packing analysis suggests a possible trimeric assembly of the SUMO‐2 protein, of which the biological significance remains to be determined.
Chloroplast phosphoglycerate kinase from Euglena gracilis
Authors: Nowitzki Ulrich , Gelius‐Dietrich Gabriel , Schwieger Maike , Henze Katrin , Martin William
Published: October 2004

Two chloroplast phosphoglycerate kinase isoforms from the photosynthetic flagellate Euglena gracilis were purified to homogeneity, partially sequenced, and subsequently cDNAs encoding phosphoglycerate kinase isoenzymes from both the chloroplast and cytosol of E. gracilis were cloned and sequenced. Chloroplast phosphoglycerate kinase, a monomeric enzyme, was encoded as a polyprotein precursor of at least four mature subunits that were separated by conserved tetrapeptides. In a Neighbor‐Net analysis of sequence similarity with homologues from numerous prokaryotes and eukaryotes, cytosolic phosphoglycerate kinase of E. gracilis showed the highest similarity to cytosolic and glycosomal homologues from the Kinetoplastida. The chloroplast isoenzyme of E. gracilis did not show a close relationship to sequences from other photosynthetic organisms but was most closely related to cytosolic homologues from animals and fungi.
17β ‐Hydroxysteroid dehydrogenase type 11 is a major peroxisome proliferator‐activated receptor α‐regulated gene in mouse intestine
Authors: Motojima Kiyoto
Published: October 2004

In order to study the role of peroxisome proliferator‐activated receptor α in mouse intestine, its agonist‐induced proteins were identified by peptide mass fingerprinting followed by Northern blot analysis using their cDNAs. One of the most remarkably induced proteins was identified as 17β‐hydroxysterol dehydrogenase type 11. Its very rapid induction by various agonists was most efficient in intestine and then in liver. These findings together with recently reported results showing the enzyme family's wide substrate spectrum, including not only glucocorticoids and sex steroids but also bile acids, fatty acids and branched chain amino acids, suggest new roles for both peroxisome proliferator‐activated receptor α and 17β‐hydroxysterol dehydrogenase type 11 in lipid metabolism and/or detoxification in the intestine.
Decreased expression of aquaporin‐5 in bleomycin‐induced lung fibrosis in the mouse
Authors: Gabazza Esteban C. , Kasper Michael , Ohta Kotsuke , Keane Michael , D’Alessandro‐Gabazza Corina , Fujimoto Hajime , Nishii Yoichi , Nakahara Hiroki , Takagi Takehiro , Menon Anil G. , Adachi Yukihiko , Suzuki Koji , Taguchi Osamu
Published: October 2004

The expression of aquaporin‐5, the major water channel expressed in alveolar, tracheal, and upper bronchial epithelium, is significantly down‐regulated during acute lung injury. In the present study, the expression of aquaporin‐5 in two different mouse models of lung fibrosis was evaluated. Lung fibrosis was induced by intratracheal and by subcutaneous infusion of bleomycin. The expression of aquaporin‐5 was investigated by immunohistochemical studies and by polymerase chain reaction. There were many cells with loss of aquaporin‐5 immunoreactivity in type I alveolar epithelial cells in the mouse models of lung fibrosis. Immunohistochemistry of lung tissue in aquaporin‐5 knockout mice revealed a fibrotic phenotype with increased deposition of extracellular collagen type I in thickened alveolar walls. Semiquantitative analysis of aquaporin‐5 mRNA expression showed more abundant content of aquaporin‐5 in the lung of the normal mouse compared to the mouse with lung fibrosis. The results of this study showed, for the first time, that chronic lung injury and lung fibrosis is associated with decreased protein and mRNA expression of aquaporin‐5 in the lung.
Combined signet ring cell and glassy cell carcinoma of the uterine cervix arising in a young Japanese woman: A case report with immunohistochemical and histochemical analyses
Authors: Moritani Suzuko , Ichihara Shu , Kushima Ryoji , Sugiura Fumiyoshi , Mushika Masafumi , Silverberg Steven G.
Published: October 2004

Signet ring cell carcinoma and glassy cell carcinoma are both rare histological subtypes of uterine cervical cancer. This report is of a case of uterine cervical carcinoma arising in a 29‐year‐old woman who had major components of signet ring cell carcinoma and glassy cell carcinoma within the same tumor. Histochemical and immunohistochemical analyses, including high and low molecular weight cytokeratins, p63 and MUC5AC, additionally demonstrated the squamous and adenocarcinomatous differentiation in the neoplastic cells, which showed otherwise unclassifiable morphology on the haematoxylin–eosin sections. A wide range of differentiation described above supports the speculation that glassy cell carcinoma may arise from the multipotential immature cells that can differentiate into both squamous and glandular cells. It would be precise to classify this tumor as adenosquamous carcinoma. Although adenosquamous carcinoma is not a rare histological subtype in the uterine cervix, it should be necessary to report the presence of glassy cells and signet ring cells when present because the presence of both components is associated with an unfavorable clinical behavior.
Coexisting Brenner tumor and struma ovarii in the right ovary: Case report and review of the literature
Authors: Yoshida Makiko , Obayashi Chiho , Tachibana Mayumi , Minami Rieko
Published: October 2004

A bilateral ovarian tumor composed of mixed Brenner tumor and struma ovarii in the right ovary and mature cystic teratoma in the left ovary, is described. Mixed Brenner tumor and struma ovarii is rare; eight cases are reviewed. In this case, in addition to the typical Brenner tumor and struma ovarii, some nests composed of both Brenner tumor and struma ovarii in one nest were found in the right ovarian tumor. Immunohistochemically, the struma ovarii is stained for thyroglobulin, and Brenner nests showed various degrees of positive stain for thyroglobulin, which is a specific finding. Brenner tumor, in this case, may produce thyroglobulin or have a receptor to thyroglobulin or analog of thyroglobulin. The origin of mixed Brenner tumor and struma ovarii may be germ cell, as described in some literature, or the Brenner tumor may be of a metaplastic nature, although the Brenner tumor is fourfold the size of struma ovarii in the case presented.