bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of the μ-opioid receptor antagonist naloxone on extinction of conditioned fear in the developing rat
Authors: Kim Jee Hyun , Richardson Rick
Published: February 2009

Several recent studies report that neurotransmitters that are critically involved in extinction in adult rats are not important for extinction in young rats. Specifically, pretest injection of the γ-aminobutryic acid (GABA) receptor inverse agonist FG7142 has no effect on extinction in postnatal day (P)17 rats, although it reverses extinction in P24 rats as reported by Kim and Richardson in an earlier paper. Further, pre-extinction injection of the N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 has no effect on extinction in P17 rats, whereas it impairs long-term extinction in P24 rats as per Langton and colleagues in an earlier work. These findings indicate that extinction in P17 rats is qualitatively different from extinction in older rats. The present study examines the involvement of the endogenous opioid system in extinction in the developing rat using systemic injections of the μ-opioid receptor antagonist naloxone. Experiment 1 showed that injection of naloxone before extinction training disrupted the acquisition of extinction in both P17 and P24 rats. This effect was dependent on central rather than peripheral μ-opioid receptors (Experiment 2), and neither pre-test nor post-extinction injection of naloxone had effects on extinction (Experiments 3 and 4). Taken together, these findings indicate that opioid neurotransmission, in contrast to GABA and NMDA activity, is critical for extinction acquisition across development.
AnArabidopsisGPI-Anchor Plasmodesmal Neck Protein with Callose Binding Activity and Potential to Regulate Cell-to-Cell Trafficking
Authors: Simpson Clare , Thomas Carole , Findlay Kim , Bayer Emmanuelle , Maule Andrew J.
Published: February 2009

Plasmodesmata (Pds) traverse the cell wall to establish a symplastic continuum through most of the plant. Rapid and reversible deposition of callose in the cell wall surrounding the Pd apertures is proposed to provide a regulatory process through physical constriction of the symplastic channel. We identified members within a larger family of X8 domain–containing proteins that targeted to Pds. This subgroup of proteins contains signal sequences for a glycosylphosphatidylinositol linkage to the extracellular face of the plasma membrane. We focused our attention on three closely related members of this family, two of which specifically bind to 1,3-β-glucans (callose) in vitro. We named this family of proteins Pd callose binding proteins (PDCBs). Yellow fluorescent protein-PDCB1 was found to localize to the neck region of Pds with potential to provide a structural anchor between the plasma membrane component of Pds and the cell wall. PDCB1, PDCB2, and PDCB3 had overlapping and widespread patterns of expression, but neither single nor combined insertional mutants for PDCB2 and PDCB3 showed any visible phenotype. However, increased expression of PDCB1 led to an increase in callose accumulation and a reduction of green fluorescent protein (GFP) movement in a GFP diffusion assay, identifying a potential association between PDCB-mediated callose deposition and plant cell-to-cell communication.
pH‐induced folding of an apoptotic coiled coil
Authors: Dutta Kaushik , Alexandrov Andrei , Huang He , Pascal Steven M.
Published: February 2009

Par‐4 is a 38‐kD protein pivotal to the apoptotic pathways of various cell types, most notably prostate cells and neurons, where it has been linked to prostate cancer and various neurodegenerative disorders including Alzheimer's and Huntington's diseases and HIV encephalitis. The C‐terminal region of Par‐4 is responsible for homodimerization and the ability of Par‐4 to interact with proposed effector molecules. In this study, we show that the C‐terminal 47 residues of Par‐4 are natively unfolded at physiological pH and temperature. Evidence is rapidly accumulating that natively unfolded proteins play an important role in various cellular functions and signaling pathways, and that folding can often be induced on complexation with effector molecules or alteration of environment. Here we use primarily CD studies to show that changes in the environment, particularly pH and temperature, can induce the Par‐4 C terminus to form a self‐associated coiled coil.
The hydrogen exchange core and protein folding
Authors: Li Renhao , Woodward Clare
Published: February 2009

A database of hydrogen‐deuterium exchange results has been compiled for proteins for which there are published rates of out‐exchange in the native state, protection against exchange during folding, and out‐exchange in partially folded forms. The question of whether the slow exchange core is the folding core (Woodward C, 1993, Trends Biochem Sci 18:359–360) is reexamined in a detailed comparison of the specific amide protons (NHs) and the elements of secondary structure on which they are located. For each pulsed exchange or competition experiment, probe NHs are shown explicitly; the large number and broad distribution of probe NHs support the validity of comparing out‐exchange with pulsed‐exchange/competition experiments. There is a strong tendency for the same elements of secondary structure to carry NHs most protected in the native state, NHs first protected during folding, and NHs most protected in partially folded species. There is not a one‐to‐one correspondence of individual NHs. Proteins for which there are published data for native state out‐exchange and ϕ values are also reviewed. The elements of secondary structure containing the slowest exchanging NHs in native proteins tend to contain side chains with high ϕ values or be connected to a turn/loop with high ϕ values. A definition for a protein core is proposed, and the implications for protein folding are discussed. Apparently, during folding and in the native state, nonlocal interactions between core sequences are favored more than other possible nonlocal interactions. Other studies of partially folded bovine pancreatic trypsin inhibitor (Barbar E, Barany G, Woodward C, 1995, Biochemistry 34:11423–11434; Barber E, Hare M, Daragan V, Barany G, Woodward C, 1998, Biochemistry 37:7822–7833), suggest that developing cores have site‐specific energy barriers between microstates, one disordered, and the other(s) more ordered.
Characterization and molecular basis of the oligomeric structure of HIV‐1 Nef protein
Authors: Arold Stefan , Hoh François , Domergue Stephanie , Birck Catherine , Delsuc Marc‐André , Jullien Magali , Dumas Christian
Published: February 2009

The Nef protein of human immunodeficiency virus type I (HIV‐1) is an important determinant for the onset of AIDS disease. The self‐association properties of HIV‐1 Nef are analyzed by chemical cross‐linking, dynamic light scattering, equilibrium analytical ultracentrifugation, and NMR spectroscopy. The experimental data show that the HIV‐1 Nef core domain forms stable homo‐dimers and trimers in solution, but not higher oligomers. These Nef homomers are not covalently linked by disulfide bridges, and the equilibrium between these forms is dependent on the Nef concentration. We further provide the molecular basis for the Nef core dimers and trimers obtained by analysis of crystallographic models. Oligomerization of biological polypeptides is a common tool used to trigger events in cellular signaling and endocytosis, both of which are targeted by Nef. The quaternary structure of Nef may be of physiological importance and may help to connect its cellular targets or to increase affinity of the viral molecule for its ligands. The herein described models for Nef dimers and trimers will allow further mutational studies to elucidate their role in vivo. These results provide novel insight into the structural and functional relationships of this important viral protein. Moreover, the oligomer interface may represent a novel target for the design of antiviral agents.
Productive and nonproductive binding to ribonuclease A: X‐ray structure of two complexes with uridylyl(2′,5′)guanosine
Authors: Vitagliano Luigi , Merlino Antonello , Zagari Adriana , Mazzarella Lelio
Published: February 2009

Guanine‐containing mono‐ and dinucleotides bind to the active site of ribonuclease A in a nonproductive mode (retro‐binding) (Aguilar CF, Thomas PJ, Mills A, Moss DS, Palmer RA, 1992, J Mol Biol 224:265‐267). Guanine binds to the highly specific pyrimidine site by forming hydrogen bonds with Thr45 and with the sulfate anion located in the P1 site. To investigate the influence of the anion present in the P1 site on retro‐binding, we determined the structure of two new complexes of RNase A with uridylyl(2′,5′)guanosine obtained by soaking two different forms of pre‐grown RNase A crystals. In one case, RNase A was crystallized without removing the sulfate anion strongly bound to the active site; in the other, the protein was first equilibrated with a basic solution to displace the anion from the P1 site. The X‐ray structures of the complexes with and without sulfate in P1 were refined using diffraction data up to 1.8 Å (R‐factor 0.192) and 2.0 Å (R‐factor 0.178), respectively. The binding mode of the substrate analogue to the protein differs markedly in the two complexes. When the sulfate is located in P1, we observe retro‐binding; whereas when the anion is removed from the active site, the uridine is productively bound at the B1 site. In the productive complex, the electron density is very well defined for the uridine moiety, whereas the downstream guanine is disordered. This finding indicates that the interactions of guanine in the B2 site are rather weak and that this site is essentially adenine preferring. In this crystal form, there are two molecules per asymmetric unit, and due to crystal packing, only the active site of one molecule is accessible to the ligand. Thus, in the same crystal we have a ligand‐bound and a ligand‐free RNase A molecule. The comparison of these two structures furnishes a detailed and reliable picture of the structural alterations induced by the binding of the substrate. These results provide structural information to support the hypotheses on the role of RNase A active site residues that have recently emerged from site‐directed mutagenesis studies.
A model of troponin‐I in complex with troponin‐C using hybrid experimental data: The inhibitory region is a β‐hairpin
Authors: Wall Michael E. , Gallagher Stephen C. , Trewhella Jill , Tung Chang‐Shung
Published: February 2009

We present a model for the skeletal muscle troponin‐C (TnC)/troponin‐I (TnI) interaction, a critical molecular switch that is responsible for calcium‐dependent regulation of the contractile mechanism. Despite concerted efforts by multiple groups for more than a decade, attempts to crystallize troponin‐C in complex with troponin‐I, or in the ternary troponin complex, have not yet delivered a high‐resolution structure. Many groups have pursued different experimental strategies, such as X‐ray crystallography, NMR, small‐angle scattering, chemical cross‐linking, and fluorescent resonance energy transfer (FRET) to gain insights into the nature of the TnC/TnI interaction. We have integrated the results of these experiments to develop a model of the TnC/TnI interaction, using an atomic model of TnC as a scaffold. The TnI sequence was fit to each of two alternate neutron scattering envelopes: one that winds about TnC in a left‐handed sense (Model L), and another that winds about TnC in a right‐handed sense (Model R). Information from crystallography and NMR experiments was used to define segments of the models. Tests show that both models are consistent with available cross‐linking and FRET data. The inhibitory region TnI (95‐114) is modeled as a flexible β‐hairpin, and in both models it is localized to the same region on the central helix of TnC. The sequence of the inhibitory region is similar to that of a β‐hairpin region of the actin‐binding protein profilin. This similarity supports our model and suggests the possibility of using an available profilin/actin crystal structure to model the TnI/actin interaction. We propose that the β‐hairpin is an important structural motif that communicates the Ca2+‐activated troponin regulatory signal to actin.
Modeling of loops in protein structures
Authors: Fiser András , Do Richard Kinh Gian , Šali Andrej
Published: February 2009

Comparative protein structure prediction is limited mostly by the errors in alignment and loop modeling. We describe here a new automated modeling technique that significantly improves the accuracy of loop predictions in protein structures. The positions of all nonhydrogen atoms of the loop are optimized in a fixed environment with respect to a pseudo energy function. The energy is a sum of many spatial restraints that include the bond length, bond angle, and improper dihedral angle terms from the CHARMM‐22 force field, statistical preferences for the main‐chain and side‐chain dihedral angles, and statistical preferences for nonbonded atomic contacts that depend on the two atom types, their distance through space, and separation in sequence. The energy function is optimized with the method of conjugate gradients combined with molecular dynamics and simulated annealing. Typically, the predicted loop conformation corresponds to the lowest energy conformation among 500 independent optimizations. Predictions were made for 40 loops of known structure at each length from 1 to 14 residues. The accuracy of loop predictions is evaluated as a function of thoroughness of conformational sampling, loop length, and structural properties of native loops. When accuracy is measured by local superposition of the model on the native loop, 100, 90, and 30% of 4–, 8–, and 12–residue loop predictions, respectively, had <2 Å RMSD error for the mainchain N, Ca, C, and O atoms; the average accuracies were 0.59 6 0.05, 1.16 6 0.10, and 2.61 6 0.16 Å, respectively. To simulate real comparative modeling problems, the method was also evaluated by predicting loops of known structure in only approximately correct environments with errors typical of comparative modeling without misalignment. When the RMSD distortion of the main‐chain stem atoms is 2.5 Å, the average loop prediction error increased by 180, 25, and 3% for 4–, 8–, and 12–residue loops, respectively. The accuracy of the lowest energy prediction for a given loop can be estimated from the structural variability among a number of low energy predictions. The relative value of the present method is gauged by (1) comparing it with one of the most successful previously described methods, and (2) describing its accuracy in recent blind predictions of protein structure. Finally, it is shown that the average accuracy of prediction is limited primarily by the accuracy of the energy function rather than by the extent of conformational sampling.
Development of a pUC19‐based recombinant plasmid to serve as a positive control in PCR for Orientia tsutsugamushi
Authors: Hanaoka Nozomu , Sakata Akiko , Takano Ai , Kawabata Hiroki , Watanabe Haruo , Kurane Ichiro , Kishimoto Toshio , Ando Shuji
Published: February 2009

A recombinant positive control plasmid was developed to be used in PCR for Orientia tsutsugamushi. This pUC19‐based plasmid contains the O. tsutsugamushi DNA sequence, part of which is replaced with Candida albicans DNA. pUC19‐Posi is a PCR control that provides several advantages over currently used positive controls for both universal and serotypical PCR.
Mitogen‐activated protein kinases are involved in tumor necrosis factor alpha production in macrophages infected with Orientia tsutsugamushi
Authors: Yun Ji‐Hyun , Koo Jung‐Eun , Koh Young‐Sang
Published: February 2009

Orientia tsutsugamushi, an obligatory intracellular bacterium, is the causative agent of scrub typhus. Here the role of MAPK in TNF‐α production in macrophages after infection with O. tsutsugamushi has been investigated. ERK1/2, JNK, and p38 MAPK became phosphorylated in Orientia‐stimulated macrophages. Selective inhibitors of MAPK cascades could all significantly reduce Orientia‐stimulated TNF‐α production. Orientia‐stimulated TNF‐α production via p38 and JNK pathways was regulated by a post‐transcriptional mechanism, whereas the ERK pathway mainly controlled the transcriptional step of TNF‐α gene expression during infection. In conclusion, our data indicate that MAPK signaling is required to induce maximal TNF‐α production in macrophages during Orientia infection.
Transient inhibition of NF‐κB by DHMEQ induces cell death of primary effusion lymphoma without HHV‐8 reactivation
Authors: Dabaghmanesh Nazanin , Matsubara Aiko , Miyake Ariko , Nakano Kazumi , Ishida Takaomi , Katano Harutaka , Horie Ryoichi , Umezawa Kazuo , Watanabe Toshiki
Published: February 2009

Primary effusion lymphoma (PEL) is a refractory malignancy caused by human herpes virus 8 (HHV‐8) in immunocompromised individuals. The tumor cells of PEL are characterized by constitutive NF‐κB activation. Dehydroxymethylepoxyquinomicin (DHMEQ) is a new NF‐κB inhibitor and is effective on various tumor cells with constitutively activated NF‐κB. Thus, in search for a new therapeutic modality of PEL, we examined the effect of DHMEQ on PEL cells. We confirmed constitutive activation of NF‐κB with subcomponents of p50 and p65 in PEL cell lines. DHMEQ quickly and transiently abrogated NF‐κB activation and reduced the cell viability in dose‐ and time‐dependent manners, inducing apoptosis through activation of both mitochondrial and membrane pathways. Array analysis revealed that DHMEQ down‐regulated expression levels of NF‐κB target genes, such asinterleukin‐6(IL6),Myc, chemokine (C‐C motif) receptor 5 (CCR5) and NF‐κB1, whereas it up‐regulated expression levels of some genes involved in apoptosis, and cell cycle arrest. DHMEQ did not reactivate HHV‐8 lytic genes, indicating that NF‐κB inhibition by DHMEQ did not induce virus replication. DHEMQ rescued CB‐17 SCID mice xenografted with PEL cells, reducing the gross appearance of effusion. Thus, DHMEQ transiently abrogated the NF‐κB activation, irreversibly triggering the apoptosis cascade without HHV‐8 reactivation. In addition, DHMEQ could rescue the PEL‐xenograft mice. Therefore, we suggest DHMEQ as a promising candidate for molecular target therapy of the PEL. (Cancer Sci2009; 100: 737–746)
Heavy‐ion‐induced bystander killing of human lung cancer cells: Role of gap junctional intercellular communication
Authors: Harada Kosaku , Nonaka Tetsuo , Hamada Nobuyuki , Sakurai Hideyuki , Hasegawa Masatoshi , Funayama Tomoo , Kakizaki Takehiko , Kobayashi Yasuhiko , Nakano Takashi
Published: February 2009

The aim of the present study was to clarify the mechanisms of cell death induced by heavy‐ion irradiation focusing on the bystander effect in human lung cancer A549 cells. In microbeam irradiation, each of 1, 5, and 25 cells under confluent cell conditions was irradiated with 1, 5, or 10 particles of carbon ions (220 MeV), and then the surviving fraction of the population was measured by a clonogenic assay in order to investigate the bystander effect of heavy‐ions. In this experiment, the limited number of cells (0.0001–0.002%, 5–25 cells) under confluent cell conditions irradiated with 5 or 10 carbon ions resulted in an exaggerated 8–14% increase in cell death by clonogenic assay. However, these overshooting responses were not observed under exponentially growing cell conditions. Furthermore, these responses were inhibited in cells treated with an inhibitor of gap junctional intercellular communication (GJIC), whereas they were markedly enhanced by the addition of a stimulator of GJIC. The present results suggest that bystander cell killing by heavy‐ions was induced mainly by direct cell‐to‐cell communication, such as GJIC, which might play important roles in bystander responses. (Cancer Sci 2009; 100: 684–688)
Silencing of SOCS1 in macrophages suppresses tumor development by enhancing antitumor inflammation
Authors: Hashimoto Masayuki , Ayada Toranoshin , Kinjyo Ichiko , Hiwatashi Kiyokazu , Yoshida Hideyuki , Okada Yasunori , Kobayashi Takashi , Yoshimura Akihiko
Published: February 2009

Inflammation has been shown to contribute to both tumor development and antitumor immunity. However, conditions determining these opposing effects are not well understood. Suppressor of cytokine signaling 1 (SOCS1) has been shown to play an important role in regulating inflammation and tumor development. It has been reported that silencing of SOCS1 gene in dendritic cells potentiates antitumor immunity, while SOCS1‐deficiency in whole organs except for T and B cells enhances inflammation‐mediated colon tumor development. To determine which types of cells are important for the suppression of tumor development by SOCS1‐deficiency, we employed the conditional knockout strategy. SOCS1 gene was deleted in macrophages and neutrophils by crossing SOCS1‐flox/flox mice with LysM‐cre mice. Resulting conditional knockout (cKO) mice showed enhanced sensitivity to endotoxin shock. SOCS1‐cKO mice survived much longer than wild‐type mice after B16 melanoma transplantation. Colon carcinogenesis induced by 1,2‐dimethylhydrazine (DMH) plus dextran sulfate sodium (DSS) was also reduced in SOCS1‐cKO mice. SOCS1‐deficiency in monocytic cells enhanced tumor‐killing activity of macrophages and tumor‐specific cytotoxic T cell activity. These results suggest that inflammation induced by SOCS1‐deficiency in monocytes potentiates antitumor immune responses rather than tumor‐promoting inflammation. (Cancer Sci 2009; 100: 730–736)
Active TGF‐β1 correlates with myofibroblasts and malignancy in the colorectal adenoma‐carcinoma sequence
Authors: Hawinkels Lukas J. A. C. , Verspaget Hein W. , Van Der Reijden Johan J. , Van Der Zon Johanna M. , Verheijen Jan H. , Hommes Daniel W. , Lamers Cornelis B. H. W. , Sier Cornelis F. M.
Published: February 2009

Transforming growth factor‐β1 (TGF‐β1), a cytokine involved in various stages of cancer, is produced as a latent complex and requires processing to become active. We have determined total and active TGF‐β1 levels in homogenates of colorectal neoplasia. In contrast to total TGF‐β levels, showing a stepwise increase in the mucosa‐adenoma‐carcinoma sequence, active TGF‐β1 levels are increased only in carcinomas but not in premalignant adenomas. Furthermore, solely active TGF‐β1 levels are associated with the stage of the carcinomas and worse patient prognosis. Active TGF‐β1 levels correlated significantly with plasminogen activator inhibitor (PAI)‐1, α‐smooth muscle actin (SMA) and several matrix‐remodeling proteinases. Interestingly, SMA levels are also significantly increased in colorectal carcinomas but not in adenomas, suggesting that despite the enhanced total TGF‐β1 levels, myofibroblast accumulation is not (yet) occurring in these premalignant neoplasias. The correlation between active TGF‐β1 and SMA expression in tumors indicates that tumor‐promoting myofibroblasts might arise as a result of increased TGF‐β1 activation. These data underline the significance of the interaction between malignant cells and (myo)‐fibroblasts in the tumor microenvironment, modulating the biologic behavior of colorectal cancer. (Cancer Sci 2009; 100: 663–670)
Immunological characterization of a Blo t 12 isoallergen: identification of immunoglobulin E epitopes
Authors: Zakzuk J. , Jiménez S. , Cheong N. , Puerta L. , Lee B. W. , Chua K. Y. , Caraballo L.
Published: February 2009

Background
Differences in the IgE response to isoallergens could have clinical implications; therefore, its analysis will contribute to the design of better strategies for the diagnosis and treatment of allergic respiratory diseases. Several isoforms have been described from mites but there is no information about the clinical impact of Blomia tropicalis isoallergens.

Objective
To evaluate the differences in the IgE response against two Blo t 12 isoallergens.

Methods
The IgE‐binding properties of Blo t 12 isoallergens were analysed by ELISA, a skin prick test and ELISA cross‐inhibition. Epitope mapping was performed using synthetic overlapping peptides. Fold recognition methods were used to model the chitin‐binding domain of the two isoallergens.

Results
The frequency and strength of the IgE response were greater for Blo t 12.0101 than for Blo t 12.0102. Three IgE‐binding areas were identified in Blo t 12.0101; one of them included two residues that are different in Blo t 12.0102. Modelling of the chitin‐binding domains of these allergens predicted that they have structural differences that could influence antibody recognition of one of these epitopes.

Conclusion
In silico structural analysis and immunological characterization of Blo t 12 reveals that allergen polymorphism influences IgE reactivity. Blo t 12.0101 is the most IgE‐reactive isoform in Cartagena. The identified IgE epitopes could be mutated to obtain hypoallergenic molecules of potential use for immunotherapy.
Production of native and modified recombinant Der p 1 molecules in tobacco plants
Authors: Burtin D. , Chabre H. , Olagnier B. , Didierlaurent A. , Couret M.‐N. , Comeau D. , Wambre E. , Laparra H. , Van Overtvelt L. , Montandon F. , Batard T. , Jonval V. , Lorphelin A. , Merle C. , Berrouet C. , Parry L. , Gomord V. , Van Ree R. , Moingeon P.
Published: February 2009

Background
As a complex molecule requiring post‐translational processing, it has been difficult to produce the Der p 1 major allergen from the Dermatophagoides pteronyssinus house dust mite in a recombinant form.

Objective
Here, we tested whether transgenic tobacco plants are suitable to express Der p 1, either as a wild‐type molecule or as variants lacking N‐glycosylation sites (Gly−) and/or cysteine protease activity (Enz−).

Methods
Using Agrobacterium tumefaciens‐based transformation, pro Der p 1 molecules bearing mutations within either the N‐glycosylation sites (N34Q, N150Q) and/or the cysteine protease‐active site (C132V) were expressed in tobacco plants. After purification by ion exchange chromatography, allergens were characterized using immunoblotting, circular dichroism (CD), as well as basophil and T lymphocyte stimulation assays.

Results
Four forms of recombinant Der p 1 (i.e. wild‐type Gly+/Enz+, as well as Gly−/Enz+, Gly+/Enz− or Gly−/Enz− variants) were successfully expressed in tobacco leaves as pro Der p 1 molecules. Spontaneous cleavage of the pro‐peptide was observed in tobacco leaf extracts for all forms of recombinant Der p 1 (r Der p 1). CD confirmed that all r Der p 1 molecules, with the exception of the Gly−/Enz− variant, exhibited secondary structures comparable to the natural protein. A cysteine protease activity was associated only with the Gly+/Enz+ form. All these molecules exhibit a profile similar to natural Der p 1 with respect to IgE immunoreactivity, basophil activation and T cell recognition.

Conclusion
A tobacco plant expression system allows the production of various forms of mature Der p 1, which could be used for diagnostic or immunotherapeutic purposes.
Characterization of regulatory T cells identified as CD4+CD25highCD39+ in patients with active tuberculosis
Authors: Chiacchio T , Casetti R , Butera O , Vanini V , Carrara S , Girardi E , Di Mitri D , Battistini L , Martini F , Borsellino G , Goletti D
Published: February 2009

Forkhead box P3 (FoxP3) is a transcription factor whose expression characterizes regulatory T cells (Treg), but it is also present on activated T cells, thus hindering correct Treg identification. Using classical markers for Treg recognition, discordant results were found in terms of Treg expansion during active tuberculosis (TB) disease. Recently CD39 has been shown to be an accurate marker for Treg detection. The objectives of this study were: (i) to identify Treg expressing CD39 in patients with TB and to compare the results with those obtained by the standard phenotypic markers; (ii) to evaluate if Treg are expanded in vitro by exogenous interleukin (IL)-2 or by antigen-specific stimulation; and (iii) to characterize Treg function on the modulation of antigen-specific responses. We enrolled 13 patients with pulmonary TB and 12 healthy controls. Treg were evaluated by flow cytometry ex vivo and after antigen-specific in vitro stimulation using CD25, FoxP3, CD127 and CD39 markers. Results indicate that CD39+ cells within the CD4+CD25high cells have Treg properties (absence of interferon-γ production and transforming growth factor-β1 release upon stimulation). Ex vivo analysis did not show significant differences between TB patients and controls of Treg by classical or novel markers. In contrast, a significantly higher percentage of Treg was found in TB patients after antigen-specific stimulation both in the presence or absence of IL-2. Depletion of CD39+ Treg increased RD1-specific responses significantly. In conclusion, CD39 is an appropriate marker for Treg identification in TB. These results can be useful for future studies to monitor Mycobacterium tuberculosis-specific response during TB.
c-Src tyrosine kinase co-associates with and phosphorylates signal transducer and activator of transcription 5b which mediates the proliferation of normal human B lymphocytes
Authors: Cayer M-P , Proulx M , Ma X-Z , Sakac D , Giguère J-F , Drouin M , Néron S , Branch D R , Jung D
Published: February 2009

c-Src is the normal human cellular protein homologue of the viral oncogene v-src. c-Src activity was reported recently to increase in CD40-activated human B lymphocytes, suggesting its involvement in proliferation. To elucidate the exact role of c-Src in this process, we investigated the effects of c-Src over-expression on normal B lymphocyte growth. B lymphocytes purified from human peripheral blood were infected with Ad5/F35 vector encoding either a constitutively active c-Src (c-Src/dominant-positive) or a dominant-negative c-Src (c-Src/DN). Little variation of B lymphocytes expansion could be observed between control enhanced yellow fluorescent protein and c-Src/dominant-positive-infected cells. In contrast, over-expression of c-Src/DN results in a 40% inhibition of B lymphocyte expansion. These results suggest that DN c-Src may compete with endogenous c-Src, resulting in partial inhibition of a transcriptional pathway involved in B lymphocyte proliferation. We demonstrate further that c-Src can phosphorylate signal transducer and activator of transcription 5b (STAT5b) on tyrosine 699 and that c-Src and STAT5b co-associate during B lymphocyte proliferation. These results confirm an important role for c-Src in the expansion of normal human B lymphocytes in vitro, in which c-Src may regulate STAT5b in the intracellular signalling pathway important for the proliferation of normal human B lymphocytes.
Lack of correlation between phenotype and genotype in untreated 21‐hydroxylase‐deficient Indonesian patients
Authors: Goossens Kristel , Juniarto Achmad Z. , Timmerman Marianna A. , Faradz Sultana M. H. , Wolffenbuttel Katja P. , Drop Stenvert L. S. , De Jong Frank H.
Published: February 2009

Background Mutations inCYP21A2lead to deficiency of 21‐hydroxylase and can have either severe or moderate effects on phenotype, which can be prevented by early treatment. We studied long‐term effects of this deficiency on phenotype in patients who had not been treated for prolonged periods and correlated these phenotypes with the mutations found in our patients.

Objective To assess the correlation between genotype and phenotype in untreated patients with 21‐hydroxylase deficiency.

Design Subjects with 21‐hydroxylase deficiency were selected from a large population of Indonesian patients with disorders of sexual differentiation.CYP21A2mutations in these patients were correlated with their phenotype in terms of genital development and steroid hormone levels.

Patients Fifteen 46,XX patients with ages between 1 and 33 years, of whom 12 had never been treated before.

Measurements Mutations inCYP21A2, genital phenotype and steroid hormone levels.

Results We found in all patientsCYP21A2mutations which affect enzyme activity, with a relatively high allele frequency of R356W (40%), I172N (20%) and IVS2 ‐ 1A > G (13%). Clitoris length was directly correlated with levels of testosterone, but not with age. The phenotype was not always concordant with the genotype: different phenotypes (mild to severe virilization) were found in sibling pairs with the mutations IVS2 ‐ 13A > G or I172N. The high frequency of homozygous mutants for R356W in patients aged from 1 to 11 years old is remarkable, as this mutation has been described only in salt‐wasting patients. In our study, this mutation caused a urogenital sinus in three out of seven cases, whereas in the remaining cases the labia were at least partially fused. This mutation caused severe virilization with remarkably high serum levels of renin. We found one novel substitution in intron 2 (IVS2 ‐ 37A > G), containing the branch site, which is likely to affect the CYP21‐enzyme. Two additional intron 2 substitutions were discovered, which are supposed to affect the 21‐hydroxylase (i.e. IVS2 + 33A > C and IVS2 + 67C > T).

Conclusion We conclude that a correlation exists between the concentration of androgens and the extent of virilization. However, there was no clear correlation between genotype and phenotype, except for the mutation R356W.
High prevalence of autonomous cortisol and aldosterone secretion from adrenal adenomas
Authors: Piaditis Georgios P. , Kaltsas Gregory A. , Androulakis Ioannis I. , Gouli Aggeliki , Makras Polyzois , Papadogias Dimitrios , Dimitriou Konstantina , Ragkou Despina , Markou Athina , Vamvakidis Kyriakos , Zografos Georgios , Chrousos Georgios
Published: February 2009

Objectives  Previous studies based on standard endocrine testing have shown a variable incidence of autonomous cortisol secretion (ACS) or autonomous aldosterone secretion (AAS) in patients with single adrenal adenomas (SAA). We tested whether the use of appropriate controls and modification of standard testing, aiming at eliminating interference from endogenous ACTH, reveals previously undetected subtle ACS and AAS by SAA.

Design  Case control study.

Patients  We investigated 151 patients with SAA and 72 matched controls with normal adrenal computerized tomography.

Measurements  All participants had arterial blood pressure recorded, and serum cortisol and aldosterone measured before and after intravenous administration of 250 μg of ACTH, and following dexamethasone administration. Eighty‐three patients and all the controls had serum aldosterone and renin measured before and after saline infusion, and after a second saline infusion following dexamethasone administration.

Results  Using the mean + 2 SD values obtained from controls after dexamethasone administration and saline infusion following dexamethasone administration, normal cut‐off values for cortisol (30·11 nm), aldosterone (67·59 pm), and aldosterone/renin ratio (9·74 pm/mU/l) were developed. Using these cut‐off values, the estimated incidence of ACS and AAS in patients with SAA was 56·63% and 24·10%, respectively, whereas 12·05% had autonomous secretion of both cortisol and aldosterone. Systolic and diastolic arterial blood pressure correlated significantly with the aldosterone/renin ratio following ΑCTH stimulation (P < 0·0002 and P < 0·001, respectively), and after saline infusion following dexamethasone administration (P < 0·003 and P < 0·002, respectively).

Conclusions  By applying new cut‐offs, ACS and AAS in patients with a SAA is very common, and aldosterone secretion correlates with arterial blood pressure.
How do you initiate oestrogen therapy in a girl who has not undergone puberty?
Authors: Hindmarsh Peter C.
Published: February 2009

The physiology of puberty needs to be taken into consideration in the induction of puberty. Puberty is a relatively slow process and replacement therapy should mimic this. Long‐term maintenance requires careful monitoring and long‐term assessment of risk‐benefit. This has not been appreciably defined in the adolescent population. Options for fertility need careful consideration and may depend on the adequacy of pubertal induction in terms of uterine development. A number of regimens are available for pubertal induction but the lack of comparisons makes it difficult to advocate for a particular regimen. There remain a number of areas of uncertainty, and future studies need to consider these issues and whether there are cardiovascular risk factor advantages to certain preparations. The long‐term risks of breast and gynaecological malignancy remain uncertain. Long‐term cohort studies are required to address these issues.
Diagnosis and quantification of factor VIII inhibitors
Authors: VERBRUGGEN B.
Published: February 2009

Summary.  The laboratory detection of factor VIII inhibitors is invariably performed by methods that measure the inactivation of factor VIII in mixtures of test plasma and exogenous factor VIII, e.g. normal pooled plasma. Unfortunately the intra‐ and inter‐laboratory variation of the inhibitor assays is rather high often resulting in unreliable results. The pH of the mixtures of test plasma and pooled plasma, incubation time and temperature, type of control sample, von Willebrand content of factor VIII deficient plasma that is used in the assay and the presence of lupus anticoagulant all influence and/or interfere with the results of inhibitor testing. In this review these assay characteristics, pitfalls and limitations of the assays are discussed.
Measurement of information and communication technology experience and attitudes to e‐learning of students in the healthcare professions: integrative review
Authors: Wilkinson Ann , While Alison E. , Roberts Julia
Published: February 2009

Title.  Measurement of information and communication technology experience and attitudes to e‐learning of students in the healthcare professions: integrative review.

Aim.  This paper is a report of a review to describe and discuss the psychometric properties of instruments used in healthcare education settings measuring experience and attitudes of healthcare students regarding their information and communication technology skills and their use of computers and the Internet for education.

Background.  Healthcare professionals are expected to be computer and information literate at registration. A previous review of evaluative studies of computer‐based learning suggests that methods of measuring learners’ attitudes to computers and computer aided learning are problematic.

Data sources.  A search of eight health and social science databases located 49 papers, the majority published between 1995 and January 2007, focusing on the experience and attitudes of students in the healthcare professions towards computers and e‐learning.

Review methods.  An integrative approach was adopted, with narrative description of findings. Criteria for inclusion were quantitative studies using survey tools with samples of healthcare students and concerning computer and information literacy skills, access to computers, experience with computers and use of computers and the Internet for education purposes.

Results.  Since the 1980s a number of instruments have been developed, mostly in the United States of America, to measure attitudes to computers, anxiety about computer use, information and communication technology skills, satisfaction and more recently attitudes to the Internet and computers for education. The psychometric properties are poorly described.

Conclusion.  Advances in computers and technology mean that many earlier tools are no longer valid. Measures of the experience and attitudes of healthcare students to the increased use of e‐learning require development in line with computer and technology advances.
Doctors’ assistants’ views of case management to improve chronic heart failure care in general practice: a qualitative study
Authors: Olbort Rebecca , Mahler Cornelia , Campbell Stephen , Reuschenbach Bernd , Müller‐Tasch Thomas , Szecsenyi Joachim , Peters‐Klimm Frank
Published: February 2009

Title.  Doctors’ assistants’ views of case management to improve chronic heart failure care in general practice: a qualitative study.

Aim.  This paper is a report of a study to explore the views, concerns and experiences of doctors’ assistants of case management for patients with chronic heart failure, while experiencing the new role of being a case manager within the Heidelberg Integrated Case Management trial.

Background.  Case management is being investigated as part of a randomised controlled trial aiming to improve care for patients with chronic systolic heart failure. In a complex, multifaceted intervention, trained doctors’ assistants (equivalent to a nursing role) adopted new tasks using standardised case management involving telephone monitoring, home visits and diagnostic screening.

Method.  In April 2007, 3 months after implementation of the intervention programme, 27 doctors’ assistants participated in four focus group interviews discussing their views on, and experiences of, case management. Thematic analysis of the data was undertaken.

Findings.  Participants believed that the most positive factors in case management were about interaction with patients, including opportunities for identifying disease and psychosocial problems. However, barriers included lack of time allocated to perform case management in addition to their normal role and poor cooperation within the practice team. According to the doctors’ assistants, the routine implementation of case management was acceptable, feasible and effective in improving the management of patients with chronic systolic heart failure.

Conclusion.  Case management enhanced the role of doctors’ assistants, leading to increased awareness of the perspective of patients with chronic disease. In the wider international primary care practice nursing context, the orchestrated delegation of tasks using specific case management may be a promising strategy for improving the quality of care of chronically ill patients and enabling patient self‐management.
Predictors of critical care admission in emergency department patients triaged as low to moderate urgency
Authors: Considine Julie , Thomas Shane , Potter Robyn
Published: February 2009

Title.  Predictors of critical care admission in emergency department patients triaged as low to moderate urgency.

Aim.  This paper is a report of a study to identify predictors of critical care admission in emergency department patients triaged as low to moderate urgency that may be apparent early in the emergency department episode of care.

Background.  Observations of clinical practice show that a number of emergency department patients triaged as low to moderate urgency require critical care admission, raising questions about the relationship between illness severity and physiological status early in the emergency department episode of care.

Methods.  A retrospective case control design was used. All participants were aged over 18 years, triaged to Australasian Triage Scale categories 3, 4 or 5, and attended emergency department between 1 July 2004 and 30 June 2005. Cases were admitted to intensive care unit or coronary care unit and controls were admitted to general medical or surgical units. Cases (n = 193) and controls (n = 193) were matched by age, gender, emergency department discharge diagnosis and triage category.

Results.  Critical care admission associated with: (i) a presenting complaint of nausea, vomiting and diarrhoea (OR = 3·40, 95%CI:1·22–9·47, P = 0·019), (ii) heart rate abnormalities at triage (OR = 2·10, 95%CI:1·19–3·71, P = 0·011), (iii) temperature abnormalities at triage (OR = 2·87 95%CI:1·05–7·89, P = 0·041), (iv) respiratory rate at first nursing assessment (OR = 1·66, 95%CI:1·05–2·06, P = 0·31) or (v) heart rate abnormalities at first nursing assessment (OR = 1·57, 95%CI = 1·04−2·39, P = 0·033).

Conclusion.  Derangements in temperature, respiratory rate and heart appear to increase risk of critical care admission. Further work using a prospective approach is needed to establish which physiological parameters have the highest predictive validity, the level(s) of physiological abnormality with highest clinical utility, and the optimal timing for collection of physiological data.
Nurse prescribing in dermatology: doctors’ and non‐prescribing nurses’ views
Authors: Stenner Karen , Carey Nicola , Courtenay Molly
Published: February 2009

Title.  Nurse prescribing in dermatology: doctors’ and non‐prescribing nurses’ views.

Aim.  This paper is a report of a study conducted to explore doctor and non‐prescribing nurse views about nurse prescribing in the light of their experience in dermatology.

Background.  The cooperation of healthcare professionals and peers is of key importance in enabling and supporting nurse prescribing. Lack of understanding of and opposition to nurse prescribing are known barriers to its implementation. Given the important role they play, it is necessary to consider how the recent expansion of nurse prescribing rights in England impacts on the views of healthcare professionals.

Method.  Interviews with 12 doctors and six non‐prescribing nurses were conducted in 10 case study sites across England between 2006 and 2007. Participants all worked with nurses who prescribed for patients with dermatological conditions in secondary or primary care. Thematic analysis was conducted on the interview data.

Results.  Participants were positive about their experiences of nurse prescribing having witnessed benefits from it, but had reservations about nurse prescribing in general. Acceptance was conditional upon the nurses’ level of experience, awareness of their own limitations and the context in which they prescribed. Fears that nurses would prescribe beyond their level of competence were expected to reduce as understanding and experience of nurse prescribing increased.

Conclusion.  Indications are that nurse prescribing can be acceptable to doctors and nurses so long as it operates within recommended parameters. Greater promotion and assessment of standards and criteria are recommended to improve understanding and acceptance of nurse prescribing.
Parenting with a diagnosis bipolar disorder
Authors: Wilson Lynere , Crowe Marie
Published: February 2009

Title.  Parenting with a diagnosis bipolar disorder.

Aim.  This paper is a report of a study of the ways in which bipolar disorder is constructed in the DSM‐IV and popular texts, and how parents who have been diagnosed as having a bipolar disorder construct their role as parent.

Background.  Research into parenting and mental illness has typically taken a deficit‐based approach that focuses on the risks to children when a parent has a mental illness. Literature that considers parenting specifically in the context of bipolar disorder retains a focus on the increased risk to their children of psychopathology or psychosocial difficulties.

Method.  A critical discourse analysis was conducted using interviews with five parents who had received a diagnosis of bipolar disorder. These interviews were examined in relation to the text that constructs the diagnosis of bipolar disorder (DSM‐IV) and the popular texts from which the parents drew their understandings of parenting.

Findings.  The need to monitor and moderate emotions was a dominant theme that emerged from the analysis. For these parents this also involved teaching moderation to their children and monitoring it in their children’s development. The consequence of this for these parents was a heightened sense of the need for self‐surveillance.

Conclusion.  The challenge for people working with parents who have been diagnosed with a bipolar disorder is to support them to feel confident in the management of their bipolar disorder and their ability to parent effectively.