bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple sequence alignment accuracy and evolutionary distance estimation
Authors: Rosenberg Michael S
Published: November 2005

Background

Sequence alignment is a common tool in bioinformatics and comparative genomics. It is generally assumed that multiple sequence alignment yields better results than pair wise sequence alignment, but this assumption has rarely been tested, and never with the control provided by simulation analysis. This study used sequence simulation to examine the gain in accuracy of adding a third sequence to a pair wise alignment, particularly concentrating on how the phylogenetic position of the additional sequence relative to the first pair changes the accuracy of the initial pair's alignment as well as their estimated evolutionary distance.

Results

The maximal gain in alignment accuracy was found not when the third sequence is directly intermediate between the initial two sequences, but rather when it perfectly subdivides the branch leading from the root of the tree to one of the original sequences (making it half as close to one sequence as the other). Evolutionary distance estimation in the multiple alignment framework, however, is largely unrelated to alignment accuracy and rather is dependent on the position of the third sequence; the closer the branch leading to the third sequence is to the root of the tree, the larger the estimated distance between the first two sequences.

Conclusion

The bias in distance estimation appears to be a direct result of the standard greedy progressive algorithm used by many multiple alignment methods. These results have implications for choosing new taxa and genomes to sequence when resources are limited.
Computational identification of strain-, species- and genus-specific proteins
Authors: Mazumder Raja , Natale Darren A , Murthy Sudhir , Thiagarajan Rathi , Wu Cathy H
Published: November 2005

Background

The identification of unique proteins at different taxonomic levels has both scientific and practical value. Strain-, species- and genus-specific proteins can provide insight into the criteria that define an organism and its relationship with close relatives. Such proteins can also serve as taxon-specific diagnostic targets.

Description

A pipeline using a combination of computational and manual analyses of BLAST results was developed to identify strain-, species-, and genus-specific proteins and to catalog the closest sequenced relative for each protein in a proteome. Proteins encoded by a given strain are preliminarily considered to be unique if BLAST, using a comprehensive protein database, fails to retrieve (with an e-value better than 0.001) any protein not encoded by the query strain, species or genus (for strain-, species- and genus-specific proteins respectively), or if BLAST, using the best hit as the query (reverse BLAST), does not retrieve the initial query protein. Results are manually inspected for homology if the initial query is retrieved in the reverse BLAST but is not the best hit. Sequences unlikely to retrieve homologs using the default BLOSUM62 matrix (usually short sequences) are re-tested using the PAM30 matrix, thereby increasing the number of retrieved homologs and increasing the stringency of the search for unique proteins. The above protocol was used to examine several food- and water-borne pathogens. We find that the reverse BLAST step filters out about 22% of proteins with homologs that would otherwise be considered unique at the genus and species levels. Analysis of the annotations of unique proteins reveals that many are remnants of prophage proteins, or may be involved in virulence. The data generated from this study can be accessed and further evaluated from the CUPID (C ore and U nique P rotein Id entification) system web site (updated semi-annually) at http://pir.georgetown.edu/cupid.

Conclusion

CUPID provides a set of proteins specific to a genus, species or a strain, and identifies the most closely related organism.
Analysis of vertebrate genomes suggests a new model for clade B serpin evolution
Authors: Kaiserman Dion , Bird Phillip I
Published: November 2005

Background

The human genome contains 13 clade B serpin genes at two loci, 6p25 and 18q21. The three genes at 6p25 all conform to a 7-exon gene structure with conserved intron positioning and phasing, however, at 18q21 there are two 7-exon genes and eight genes with an additional exon yielding an 8-exon structure. Currently, it is not known how these two loci evolved, nor which gene structure arose first – did the 8-exon genes gain an exon, or did the 7-exon genes lose one? Here we use the genomes of diverse vertebrate species to plot the emergence of clade B serpin genes and to identify the point at which the two genomic structures arose.

Results

Analysis of the chicken genome indicated the presence of a single clade B serpin gene locus, containing orthologues of both human loci and both genomic structures. The frog genome and the genomes of three fish species presented progressively simpler loci, although only the 7-exon structure could be identified. The Serpinb12 gene contains seven exons in the frog genome, but eight exons in chickens and humans, indicating that the additional exon evolved in this gene.

Conclusion

We propose a new model for clade B serpin evolution from a single 7-exon gene (either Serpinb1 or Serpinb6). An additional exon was gained in the Serpinb12 gene between the tetrapoda and amniota radiations to produce the 8-exon structure. Both structures were then duplicated at a single locus until a chromosomal breakage occurred at some point along the mammalian lineage resulting in the two modern loci.
A truncated Kv1.1 protein in the brain of the megencephalymouse: expression and interaction
Authors: Persson Ann-Sophie , Klement Göran , Almgren Malin , Sahlholm Kristoffer , Nilsson Johanna , Petersson Susanna , Århem Peter , Schalling Martin , Lavebratt Catharina
Published: November 2005

Background

The megencephaly mouse, mceph/mceph, is epileptic and displays a dramatically increased brain volume and neuronal count. The responsible mutation was recently revealed to be an eleven base pair deletion, leading to a frame shift, in the gene encoding the potassium channel Kv1.1. The predicted MCEPH protein is truncated at amino acid 230 out of 495. Truncated proteins are usually not expressed since nonsense mRNAs are most often degraded. However, high Kv1.1 mRNA levels in mceph/mceph brain indicated that it escaped this control mechanism. Therefore, we hypothesized that the truncated Kv1.1 would be expressed and dysregulate other Kv1 subunits in the mceph/mceph mice.

Results

We found that the MCEPH protein is expressed in the brain of mceph/mceph mice. MCEPH was found to lack mature (Golgi) glycosylation, but to be core glycosylated and trapped in the endoplasmic reticulum (ER). Interactions between MCEPH and other Kv1 subunits were studied in cell culture, Xenopus oocytes and the brain. MCEPH can form tetramers with Kv1.1 in cell culture and has a dominant negative effect on Kv1.2 and Kv1.3 currents in oocytes. However, it does not retain Kv1.2 in the ER of neurons.

Conclusion

The megencephaly mice express a truncated Kv1.1 in the brain, and constitute a unique tool to study Kv1.1 trafficking relevant for understanding epilepsy, ataxia and pathologic brain overgrowth.
Substance P selectively decreases paired pulse depression in the rat hippocampal slice
Authors: Wease Kerrie N , Davies Stephen N
Published: November 2005

Background

Although being widespread in the hippocampus, the role tachykinins play in synaptic transmission is unclear. The effect of substance P on field potentials evoked by stimulation of the Schaffer collateral-commissural fibres and recorded from the CA1 region of the rat hippocampal slice were studied.

Results

Perfusion of substance P (8 μM) had no effect on the fEPSP or population spike. Substance P did however cause a selective reduction in the paired pulse depression of population spikes evoked by paired stimulation at interpulse intervals of 20–80 msec. A comparison of the actions of other tachykinin receptor agonists gave an order of potency of substance P > [β-Ala8]-neurokinin A (4–10) > senktide. The effect of substance P was reduced by the neurokinin-1 receptor antagonist SR140333, but not by the neurokinin-2 or neurokinin-3 receptor antagonists, MDL 29,913 or [Trp7, β-Ala8]-neurokinin A (4–10).

Conclusion

The order of potency of the agonists, and the effects of the antagonists, both indicate that the effect of substance P on paired pulse depression is mediated by neurokinin-1 receptors.
Comparative assessment of three different indices of multimorbidity for studies on health-related quality of life
Authors: Fortin Martin , Hudon Catherine , Dubois Marie-France , Almirall José , Lapointe Lise , Soubhi Hassan
Published: November 2005

Background

Measures of multimorbidity are often applied to source data, populations or outcomes outside the scope of their original developmental work. As the development of a multimorbidity measure is influenced by the population and outcome used, these influences should be taken into account when selecting a multimorbidity index. The aim of this study was to compare the strength of the association of health-related quality of life (HRQOL) with three multimorbidity indices: the Cumulative Illness Rating Scale (CIRS), the Charlson index (Charlson) and the Functional Comorbidity Index (FCI). The first two indices were not developed in light of HRQOL.

Methods

We used data on chronic diseases and on the SF-36 questionnaire assessing HRQOL of 238 adult primary care patients who participated in a previous study. We extracted all the diagnoses for every patient from chart review to score the CIRS, the FCI and the Charlson. Data for potential confounders (age, sex, self-perceived economic status and self-perceived social support) were also collected. We calculated the Pearson correlation coefficients (r) of the SF-36 scores with the three measures of multimorbidity, as well as the coefficient of determination, R2, while controlling for confounders.

Results

The r values for the CIRS (range: -0.55 to -0.18) were always higher than those for the FCI (-0.47 to -0.10) and Charlson (-0.31 to -0.04) indices. The CIRS explained the highest percent of variation in all scores of the SF-36, except for the Mental Component Summary Score where the variation was not significant. Variations explained by the FCI were significant in all scores of SF-36 measuring physical health and in two scales evaluating mental health. Variations explained by the Charlson were significant in only three scores measuring physical health.

Conclusion

The CIRS is a better choice as a measure of multimorbidity than the FCI and the Charlson when HRQOL is the outcome of interest. However, the FCI may provide a good option to evaluate the physical aspect of HRQOL for the ease in its administration and scoring. The Charlson index may not be recommended as a measure of multimorbidity in studies related to either physical or mental aspects of HRQOL.
Recent developments in targeting access to high cost medicines in Australia
Authors: Lu Christine Y , Ritchie Jan , Williams Kenneth M , Day Richard O
Published: November 2005

Background

In Australia, the Pharmaceutical Benefits Scheme (PBS) has developed a set of arrangements to control access to high-cost medicines to ensure their use is cost-effective. These medicines include the tumour necrosis factor-alpha inhibitors (TNFIs) for the treatment of rheumatoid arthritis. The aim of this first phase of a qualitative study was to explore basic views on the restricted access to TNFIs in order to confirm where further investigation should take place in the next phase.

Methods

Semi-structured interviews were conducted in 2004 with a member of the four relevant stakeholder groups. Participants were asked their opinions about features of the establishment, process and effects of the system of restricted access to TNFIs. Views on the collaboration between stakeholder groups in the decision-making process were also collected.

Results

The principle of 'controlled access' to TNFIs was supported in general. There were concerns regarding some of the specific eligibility criteria. Wider and more transparent stakeholder consultation was judged desirable. Some flexibility around prescribing of TNFIs by physicians, and regular review of the arrangements were proposed. These themes will inform the next phase of the study.

Conclusion

This first phase highlighted a range of issues associated with the PBS arrangements restricting access to TNFIs. Timely review and report of issues and concerns associated with such policy developments that arose in practice are essential. There is a need for a more comprehensive exploration across a wide range of stakeholders with different perspectives that will in turn be helpful in guiding policy and practice around national arrangements to manage access to high-cost medicines.
ASIC3, an Acid-Sensing Ion Channel, is Expressed in Metaboreceptive Sensory Neurons
Authors: Molliver Derek C , Immke David C , Fierro Leonardo , Paré Michel , Rice Frank L , McCleskey Edwin W
Published: November 2005

Background:

ASIC3, the most sensitive of the acid-sensing ion channels, depolarizes certain rat sensory neurons when lactic acid appears in the extracellular medium. Two functions have been proposed for it: 1) ASIC3 might trigger ischemic pain in heart and muscle; 2) it might contribute to some forms of touch mechanosensation. Here, we used immunocytochemistry, retrograde labelling, and electrophysiology to ask whether the distribution of ASIC3 in rat sensory neurons is consistent with either of these hypotheses.

Results:

Less than half (40%) of dorsal root ganglion sensory neurons react with anti-ASIC3, and the population is heterogeneous. They vary widely in cell diameter and express different growth factor receptors: 68% express TrkA, the receptor for nerve growth factor, and 25% express TrkC, the NT3 growth factor receptor. Consistent with a role in muscle nociception, small (<25 μm) sensory neurons that innervate muscle are more likely to express ASIC3 than those that innervate skin (51% of small muscle afferents vs. 28% of small skin afferents). Over 80% of ASIC3+ muscle afferents co-express CGRP (a vasodilatory peptide). Remarkably few (9%) ASIC3+ cells express P2X3 receptors (an ATP-gated ion channel), whereas 31% express TRPV1 (the noxious heat and capsaicin-activated ion channel also known as VR1). ASIC3+/CGRP+ sensory nerve endings were observed on muscle arterioles, the blood vessels that control vascular resistance; like the cell bodies, the endings are P2X3- and can be TRPV1+. The TrkC+/ASIC3+ cell bodies are uniformly large, possibly consistent with non-nociceptive mechanosensation. They are not proprioceptors because they fail two other tests: ASIC3+ cells do not express parvalbumin and they are absent from the mesencephalic trigeminal nucleus.

Conclusion:

Our data indicates that: 1) ASIC3 is expressed in a restricted population of nociceptors and probably in some non-nociceptors; 2) co-expression of ASIC3 and CGRP, and the absence of P2X3, are distinguishing properties of a class of sensory neurons, some of which innervate blood vessels. We suggest that these latter afferents may be muscle metaboreceptors, neurons that sense the metabolic state of muscle and can trigger pain when there is insufficient oxygen.
Influence of static lumbar flexion on the trunk muscles' response to sudden arm movements
Authors: Lehman Gregory J , Story Stephen , Mabee Robert
Published: November 2005

Background

Viscoelastic creep of lumbar ligaments (prolonged forward bend) has been shown to negatively influence the spine's muscular reflexive behaviour and spinal stability. No studies to date have investigated the influence of spinall viscoelastic creep on the feedforward response of the trunk muscles to sudden arm raises.

Methods

Surface myoelectric activity was collected from the transversus abdominis/internal oblique, the lower erector spinae and the deltoid muscle during sudden ballistic arm raising before and after 10 minutes of prolonged forward bend in 11 healthy participants free of low back injury. The timing of trunk muscle activity relative to the deltoid muscle was calculated for 5 trials before and 5 trials after the creep procedure.

Results

Viscoelastic creep had no influence on the feedforward response of the trunk muscles during sudden arm raises. A feedforward response of the trunk muscles was not seen in every study participant and during every trial.

Conclusion

Passive trunk muscle fatigue does not appear to influence the timing of the stabilizing role of the investigated trunk muscles to sudden arm flexion.
Facilitating follow-up in ELT INSET
Authors: Waters Alan
Published: November 2005

There is evidence that ELT INSET does not always result in the desired level of ‘follow-up’, i.e. impact on teachers’ classroom practices. Nevertheless, little research appears to have been carried out concerning how the design of INSET systems affects such outcomes. This paper therefore attempts to throw light on some of the factors involved, using data derived from research into the operation of the Philippines English Language Teaching (PELT) Project INSET system. In order to attempt to facilitate follow-up, this programme took the form of a hybrid, joint course- and school-based model. Trainees’ views on the functioning of the model were elicited via questionnaires and structured discussions. The findings show that the following variables are among those which are important in determining how effectively such an INSET system operates: 1) the nature of its course- based component; 2) the interface between trainees, trainers and school- based ‘ELT managers’; and 3) the form of school-based follow-up activity employed. The data also indicates how these elements might be configured so as to optimize the potential for INSET follow-up to occur.
How can insights from conversation analysis be directly applied to teaching L2 pragmatics?
Authors: Huth Thorsten , Taleghani-Nikazm Carmen
Published: November 2005

This paper revisits the question of why pragmatics should be taught in the foreign language classroom and demonstrates how this can be achieved effectively with materials informed by conversation analysis (CA). Since findings in CA describe systematic action sequences underlying verbal activities that display cross-cultural variation, they capture pragmatics in its most natural locus: the conversational encounter. It will furthermore be demonstrated that L2 learners may benefit from instruction with CA-based materials with the ability to anticipate, interpret and produce, socio-pragmatically appropriate verbal behaviour in the target language. CA-based materials thus provide a rich resource for language teachers based on solid empirical evidence, and effectively enable L2 learners to engage in cross-culturally variable language behaviour inside and outside of class.
Transgenic Mice Expressing P450 Aromatase as a Model for Male Infertility Associated with Chronic Inflammation in the Testis
Authors: Li Xiangdong , Strauss Leena , Kaatrasalo Annukka , Mayerhofer Artur , Huhtaniemi Ilpo , Santti Risto , Mäkelä Sari , Poutanen Matti
Published: November 2005

Our previous studies have shown that transgenic male mice expressing human P450 aromatase (AROM+) are infertile. In the present study, we followed the testis phenotype up to 15 months of age in these mice. The testes of the old AROM+ mice showed Leydig cell hypertrophy and hyperplasia, as indicated by the staining for steroidogenic enzymes and androgen and estrogen receptors. However, the Leydig cell adenomas did not show signs of malignization. In contrast, we observed a marked increase in the number of activated macrophages in the testicular interstitium of the aging AROM+ mice. The macrophages were further shown to express high levels of CD68 (a monocyte/macrophage marker) and secrete TNFα, indicating strong activation, presumably by estrogen exposure. The increased activity of the macrophages was associated with Leydig cell depletion (analyzed at the age of 9 and 15 months) and an increased number of mast cells and fibrosis in the testicular interstitium. Interestingly, similar findings have been made in testes of infertile men. Hence, the aging AROM+ males present with a phenocopy of inflammation-associated infertility in men, providing a model for further studies on the putative link among estrogens, orchitis, and infertility.
Effects of Rexinoids on Thyrotrope Function and the Hypothalamic-Pituitary-Thyroid Axis
Authors: Sharma Vibha , Hays William R. , Wood William M. , Pugazhenthi Umarani , St. Germain Donald L. , Bianco Antonio C. , Krezel Wojciech , Chambon Pierre , Haugen Bryan R.
Published: November 2005

Retinoid X receptor (RXR)-selective retinoids (rexinoids) can cause central hypothyroidism in humans, and this effect has been confirmed in rodent models. In this report, we characterized the effect of rexinoids on the hypothalamic-pituitary-thyroid axis in mice and TSH regulation in a thyrotrope-derived cell line. The synthetic rexinoid (LG 268) suppressed TSH and T4 levels in mice. Hypothalamic TRH mRNA was unaffected, but steady-state pituitary TSHβ mRNA levels were significantly lowered, suggesting a direct effect of rexinoids on thyrotropes. LG 268 suppressed TSH protein secretion and TSHβ mRNA in TαT1 thyrotropes as early as 8 h after treatment, whereas the retinoic acid receptor-selective retinoid (TTNPB) had no effect. Type 2 iodothyronine deiodinase (D2) mRNA and activity were suppressed by LG 268 in TαT1 cells, whereas only D2 mRNA was suppressed in mouse pituitaries. LG 268 suppressed TSHβ promoter activity by 42% and the −200 to −149 region accounted for a majority of the LG 268-mediated suppression of promoter activity. The RXRγ isotype is expressed in thyrotropes. In vitro transfection and in vivo transgenic studies indicate that any RXR isotype can mediate TSH suppression by rexinoids, but the RXRγ isotype is most efficient at mediating this response. RXRγ-deficient mice lacked pituitary D2 mRNA suppression by LG 268, but D2 activity remained intact. In summary, RXR-selective retinoids (rexinoids) have multiple effects on the hypothalamic-pituitary-thyroid axis. Rexinoids directly suppress TSH secretion, TSHβ mRNA levels and promoter activity, and D2 mRNA levels but have no direct effect on hypothalamic TRH levels. Rexinoids also stimulate type 1 iodothyronine deiodinase activity in the liver and pituitary.
Neutrophils and Macrophages Promote Angiogenesis in the Early Stage of Endometriosis in a Mouse Model
Authors: Lin Yiu-Jiuan , Lai Ming-Derg , Lei Huan-Yao , Wing Lih-Yuh C.
Published: November 2005

Substantial evidence suggests that inflammatory cytokines, immune cells, and angiogenesis are important for endometriosis. In this study, we investigated the role of the sequential events in the development of endometriosis in a mouse model. Uterine tissue was transplanted into the peritoneum of ovariectomized mice and then supplemented with estrogen or vehicle. On different days after transplantation, cell proliferation, angiogenesis, and infiltrated immune cells in ectopic tissue were examined using immunochemical staining. Many disintegrated blood vessels but no bromodeoxyuridine-positive cells in ectopic tissue were observed in the estrogen-treated group on posttransplantation d 1 and 2. On d 4–7, bromodeoxyuridine-positive cells were detected in the blood vessels of ectopic tissue, indicating that angiogenesis was initiated in this stage. Angiogenesis also occurred in ectopic tissue in the vehicle-treated group. Profound infiltration of neutrophils in ectopic tissue occurred on d 1–4, when the number of neutrophils and levels of macrophage inflammatory protein (MIP)-1α and MIP-2 chemokines in peritoneal fluids also reached their peak. Peritoneal macrophage numbers did not change, but secretions of TNFα, IL-6, MIP-1α, and MIP-2 from macrophages isolated on d 2 were higher than on d 0. In vitro studies showed that peritoneal neutrophils and macrophages secreted vascular endothelial growth factor, which was up-regulated by TNFα and IL-6. Our results suggest that neutrophils and macrophages may promote angiogenesis in the early stage of endometriosis and that chemokines and cytokines amplify the angiogenic signal for the growth of endometriotic tissue.
Activity-Dependent Potentiation of Large Dense-Core Vesicle Release Modulated by Mitogen-Activated Protein Kinase/Extracellularly Regulated Kinase Signaling
Authors: Park Yong-Soo , Jun Dong-Jae , Hur Eun-Mi , Lee Sun-Kyong , Suh Byung-Sun , Kim Kyong-Tai
Published: November 2005

Large dense-core vesicles (LDCVs), containing neuropeptides, hormones, and amines, play a crucial role in the activation of the sympathetic nervous system and synaptic modulation. In some secretory cells, LDCVs show activity-dependent potentiation (ADP), which represents enhancement of subsequent exocytosis, compared with the previous one. Here we report the signaling mechanism involved in ADP of LDCV release. First, ADP of LDCV release, induced by repetitive stimulation of nicotinic acetylcholine receptors (nAChRs), was augmented by increasing calcium influx, showing calcium dependence of ADP. Second, translocation of vesicles was involved in ADP. Electron microscope analysis revealed that nAChR stimulation resulted in LDCV translocation to the plasma membrane and increase of fused LDCVs in response to repetitive stimulation was observed by amperometry. Third, we provide evidence for involvement of MAPK signaling in ADP. MAPK signaling was activated by nAChR-induced calcium influx, and ADP as well as vesicle translocation was suppressed by inhibition of MAPK signaling with MAPK kinase blockers, such as PD 098059 and U0126. Fourth, PD 098059 inhibited nAChR stimulation-induced F-actin disassembly, which has been reported to control vesicle translocation. Taken together, we suggest that ADP of LDCV release is modulated by calcium-dependent activation of MAPK signaling via regulating F-actin disassembly.
Estrogen Induction of the Granzyme B Inhibitor, Proteinase Inhibitor 9, Protects Cells against Apoptosis Mediated by Cytotoxic T Lymphocytes and Natural Killer Cells
Authors: Jiang Xinguo , Orr Brent A. , Kranz David M. , Shapiro David J.
Published: November 2005

Exposure to estrogens is associated with an increased risk of developing breast, cervical, and liver cancer. Estrogens strongly induce the human granzyme B inhibitor, proteinase inhibitor 9 (PI-9). Because cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the granzyme pathway to induce apoptosis of target cells, we tested the ability of activated CTLs and the human NK cell line, YT cells, to lyse human liver cells. Estrogen induction of PI-9 protected the liver cells against CTL and NK cell-mediated, granzyme-dependent, apoptosis. Knockdown of PI-9 by RNA interference blocked the protective effect of estrogen. This work demonstrates that estrogens can act on target cells to control their destruction by immune system cells and shows that induction of PI-9 expression can inhibit both CTL and NK cell-mediated apoptosis. Estrogen induction of PI-9 may reduce the ability of cytolytic lymphocytes-mediated immune surveillance to destroy newly transformed cells, possibly providing a novel mechanism for an estrogen-mediated increase in tumor incidence.
Molecular Phenotyping of Human Endometrium Distinguishes Menstrual Cycle Phases and Underlying Biological Processes in Normo-Ovulatory Women
Authors: Talbi S. , Hamilton A. E. , Vo K. C. , Tulac S. , Overgaard M. T. , Dosiou C. , Le Shay N. , Nezhat C. N. , Kempson R. , Lessey B. A. , Nayak N. R. , Giudice L. C.
Published: November 2005

Histological evaluation of endometrium has been the gold standard for clinical diagnosis and management of women with endometrial disorders. However, several recent studies have questioned the accuracy and utility of such evaluation, mainly because of significant intra- and interobserver variations in histological interpretation. To examine the possibility that biochemical or molecular signatures of endometrium may prove to be more useful, we have investigated whole-genome molecular phenotyping (54,600 genes and expressed sequence tags) of this tissue sampled across the cycle in 28 normo-ovulatory women, using high-density oligonucleotide microarrays. Unsupervised principal component analysis of all samples revealed that samples self-cluster into four groups consistent with histological phenotypes of proliferative (PE), early-secretory (ESE), mid-secretory (MSE), and late-secretory (LSE) endometrium. Independent hierarchical clustering analysis revealed equivalent results, with two major dendrogram branches corresponding to PE/ESE and MSE/LSE and sub-branching into the four respective phases with heterogeneity among samples within each sub-branch. K-means clustering of genes revealed four major patterns of gene expression (high in PE, high in ESE, high in MSE, and high in LSE), and gene ontology analysis of these clusters demonstrated cycle-phase-specific biological processes and molecular functions. Six samples with ambiguous histology were identically assignable to a cycle phase by both principal component analysis and hierarchical clustering. Additionally, pairwise comparisons of relative gene expression across the cycle revealed genes/families that clearly distinguish the transitions of PE→ESE, ESE→MSE, and MSE→LSE, including receptomes and signaling pathways. Select genes were validated by quantitative RT-PCR. Overall, the results demonstrate that endometrial samples obtained by two different sampling techniques (biopsy and curetting hysterectomy specimens) from subjects who are as normal as possible in a human study and including those with unknown histology, can be classified by their molecular signatures and correspond to known phases of the menstrual cycle with identical results using two independent analytical methods. Also, the results enable global identification of biological processes and molecular mechanisms that occur dynamically in the endometrium in the changing steroid hormone milieu across the menstrual cycle in normo-ovulatory women. The results underscore the potential of gene expression profiling for developing molecular diagnostics of endometrial normalcy and abnormalities and identifying molecular targets for therapeutic purposes in endometrial disorders.
Cyclic Adenosine 5′-Monophosphate-Dependent Sphingosine-1-Phosphate Biosynthesis Induces Human CYP17 Gene Transcription by Activating Cleavage of Sterol Regulatory Element Binding Protein 1
Authors: Ozbay Tuba , Rowan Anne , Leon Adam , Patel Pritty , Sewer Marion B.
Published: November 2005

In the human adrenal cortex, ACTH activates steroid hormone biosynthesis by acutely increasing cholesterol delivery to the mitochondrion and chronically increasing the transcription of steroidogenic genes (including CYP17) via a cAMP-dependent pathway. In the present study, we characterized the role of sphingolipids in ACTH-dependent steroidogenesis. H295R human adrenocortical cells were treated with ACTH or dibutyryl cAMP (Bt2cAMP) and the content of several sphingolipid species quantified by mass spectrometry. Both ACTH and Bt2cAMP decreased cellular amounts of several sphingolipids, including sphingomyelin, ceramides, and sphingosine and stimulating the activity of sphingosine kinase and increasing the release of sphingosine-1-phosphate (S1P) into the media. S1P increased CYP17 mRNA expression by promoting the cleavage and nuclear localization of sterol regulatory element binding protein (SREBP) 1. Chromatin immunoprecipitation assays revealed that Bt2cAMP and S1P increased acetylation of histone H3 and promoted binding of SREBP1 to the −520/−331 region of the CYP17 promoter. In summary, our studies demonstrate a role for sphingolipid metabolism and SREBP1 in ACTH-dependent CYP17 regulation and steroidogenesis.
Recruitment of the Tyrosine Phosphatase Src Homology 2 Domain Tyrosine Phosphatase-2 to the p85 Subunit of Phosphatidylinositol-3 (PI-3) Kinase Is Required for Insulin-Like Growth Factor-I-Dependent PI-3 Kinase Activation in Smooth Muscle Cells
Authors: Kwon Mijin , Ling Yan , Maile Laura A. , Badley-Clark Jane , Clemmons David R.
Published: November 2005

IGF-I stimulates smooth muscle cell (SMC) migration and the phosphatidylinositol-3 (PI-3) kinase pathway plays an important role in mediating the IGF-I-induced migratory response. Prior studies have shown that the tyrosine phosphatase Src homology 2 domain tyrosine phosphatase (SHP)-2 is necessary to activate PI-3 kinase in response to growth factors and expression of a phosphatase inactive form of SHP-2 (SHP-2/C459S) impairs IGF-I-stimulated cell migration. However, the mechanism by which SHP-2 phosphatase activity or the recruitment of SHP-2 to other signaling molecules contributes to IGF-I stimulated PI-3 kinase activation has not been determined. SMCs that had stable expression of SHP-2/C459S had reduced cell migration and Akt activation in response to IGF-I, compared with SMC-expressing native SHP-2. Similarly in cells expressing native SHP-2, IGF-I induced SHP-2 binding to p85, whereas in cells expressing SHP-2/C459S, there was no increase. Because the C459S substitution results in loss of the ability of SHP-2 to disassociate from its substrates, making it inaccessible not only to p85 but also the other proteins, a p85 mutant in which tyrosines 528 and 556 were changed to phenylalanines was prepared to determine whether this would disrupt the p85/SHP-2 interaction and whether the loss of this specific interaction would alter IGF-I stimulated the cell migration. Substitution for these tyrosines in p85 resulted in loss of SHP-2 recruitment and was associated with a reduction in association of the p85/p110 complex with insulin receptor substrate-1. Cells stably expressing this p85 mutant also showed a decrease in IGF-I-stimulated PI-3 kinase activity and cell migration. Preincubation of cells with a cell-permeable peptide that contains the tyrosine556 motif of p85 also disrupted SHP-2 binding to p85 and inhibited the IGF-I-induced increase in cell migration. The findings indicate that tyrosines 528 and 556 in p85 are required for SHP-2 association. SHP-2 recruitment to p85 is required for IGF-I-stimulated association of the p85/p110 complex with insulin receptor substrate-1 and for the subsequent activation of the PI-3 kinase pathway leading to increased cell migration.
The Loss of the Chloride Channel, ClC-5, Delays Apical Iodide Efflux and Induces a Euthyroid Goiter in the Mouse Thyroid Gland
Authors: van den Hove Marie-France , Croizet-Berger Karine , Jouret François , Guggino Sandra E. , Guggino William B. , Devuyst Olivier , Courtoy Pierre J.
Published: November 2005

Genetic inactivation of ClC-5, a voltage-gated chloride channel prominently expressed in the kidney, leads to proteinuria because of defective apical endocytosis in proximal tubular cells. Because thyroid hormone secretion depends on apical endocytosis of thyroglobulin (Tg), we investigated whether ClC-5 is expressed in the thyroid and affects its function, using Clcn5-deficient knockout (KO) mice. We found that ClC-5 is highly expressed in wild-type mouse thyroid (∼40% of mRNA kidney level). The protein was immunolocalized at the apical pole of thyrocytes. In Percoll gradients, ClC-5 overlapped with plasma membrane and early endosome markers, but best codistributed with the late endosomal marker, Rab7. ClC-5 KO mice were euthyroid (normal T4 and TSH serum levels) but developed a goiter with parallel iodine and Tg accumulation (i.e. normal Tg iodination level). When comparing ClC-5 KO with wild-type mice, thyroid 125I uptake after 1 h was doubled, incorporation into Tg was decreased by approximately 2-fold, so that trichloroacetic acid-soluble 125I increased approximately 4-fold. Enhanced 125I− efflux upon perchlorate and presence of 125I-Tg as autoradiographic rings at follicle periphery demonstrated delayed iodide organification. Endocytic trafficking of 125I-Tg toward lysosomes was not inhibited. Expression of pendrin, an I−/Cl− exchanger involved in apical iodide efflux, was selectively decreased by 60% in KO mice at mRNA and protein levels. Thus, ClC-5 is well expressed in the thyroid but is not critical for apical endocytosis, contrary to the kidney. Instead, the goiter associated with ClC-5 KO results from impaired rate of apical iodide efflux by down-regulation of pendrin expression.
Estrogen Dendrimer Conjugates that Preferentially Activate Extranuclear, Nongenomic Versus Genomic Pathways of Estrogen Action
Authors: Harrington William R. , Kim Sung Hoon , Funk Cory C. , Madak-Erdogan Zeynep , Schiff Rachel , Katzenellenbogen John A. , Katzenellenbogen Benita S.
Published: November 2005

Estrogenic hormones are classically thought to exert their effects by binding to nuclear estrogen receptors and altering target gene transcription, but estrogens can also have nongenomic effects through rapid activation of membrane-initiated kinase cascades. The development of ligands that selectively activate only the nongenomic pathways would provide useful tools to investigate the significance of these pathways. We have prepared large, abiotic, nondegradable poly(amido)amine dendrimer macromolecules that are conjugated to multiple estrogen molecules through chemically robust linkages. Because of their charge and size, these estrogen-dendrimer conjugates (EDCs) remain outside the nucleus. They stimulate ERK, Shc, and Src phosphorylation in MCF-7 breast cancer cells at low concentrations, yet they are very ineffective in stimulating transcription of endogenous estrogen target genes, being approximately 10,000-fold less potent than estradiol in genomic actions. In contrast to estradiol, EDC was not effective in stimulating breast cancer cell proliferation. Because these EDC ligands activate nongenomic activity at concentrations at which they do not alter the transcription of estrogen target genes, they should be useful in studying extranuclear initiated pathways of estrogen action in a variety of target cells.
Western Blotting of Total Lysate of Helicobacter pylori in Cases of Atrophic Body Gastritis
Authors: Mini Roberta , Annibale Bruno , Lahner Edith , Bernardini Giulia , Figura Natale , Santucci Annalisa
Published: November 2005

Background: Atrophic body gastritis is considered the first important step in the histogenesis of gastric carcinoma, a multistep process starting from chronic gastritis and progressing through chronic atrophic gastritis, intestinal metaplasia, and dysplasia. Helicobacter pylori is involved in the induction of atrophic body gastritis, but documentation of H. pylori infection is difficult because of the progressive disappearance of the bacterium. Our study aimed to detect past H. pylori infection in patients with atrophic body gastritis.

Methods: We used Western blot analyses of whole bacterial protein lysate of 2 different strains to probe sera from 143 patients. All sera were analyzed by ELISA (Bio-Rad), and results of gastric histology were available for all patients.

Results: Among 111 patient sera previously classified as negative for H. pylori infection by ELISA, 106 (95.5%) were positive when assayed by immunoblotting.

Conclusions: Commercial diagnostic reagent sets may fail to detect H. pylori infection. Western blotting of whole bacterial protein extracts could provide the basis of a noninvasive serology tool able to assess previous infection with H. pylori in patients with atrophic body gastritis.
Feasibility Study of New Calibrators for Thyroid-Stimulating Hormone (TSH) Immunoprocedures Based on Remodeling of Recombinant TSH to Mimic Glycoforms Circulating in Patients with Thyroid Disorders
Authors: Donadio Sandrine , Pascual Aurélie , Thijssen Jos HH , Ronin Catherine
Published: November 2005

Background: Differences between the glycosylation patterns of a pituitary thyroid-stimulating hormone calibrator (pitTSH) and serum samples have been shown to be responsible for nonidentical epitope expression and for introducing discrepancies in TSH measurements. We studied the feasibility of developing new candidate reference materials by remodeling recombinant TSH (recTSH) to generate potential mimics of serum TSH.

Methods: Terminal sialylation and/or inner fucosylation of recTSH were remodeled by a combination of enzyme treatments followed (or not) by lentil lectin-Sepharose affinity chromatography. The resulting TSH preparations were screened for epitope similarity in 23 immunoassays mapping 3 antigenic clusters common to the pitTSH 2nd International Reference Preparation (IRP) and the recTSH 1st IRP and then challenged against a pool of 63 patients with increased serum TSH (>60 mIU/L).

Results: pitTSH was poorly correlated with serum TSH, with a mean (SD) slope of 2.124 (0.001), in contrast to recTSH [slope, 1.178 (0.056)]. Comparison of variably sialylated preparations with recTSH gave slopes of 0.860 (0.057) for desialylated TSH, 1.064 (0.057) for α2,3/6-oversialylated recTSH, and 0.953 (0.033) for α2,6-resialylated recTSH, indicating that TSH forms enriched in sialic acid closely resemble serum TSH. Further testing against serum TSH showed satisfactory agreement with both TSH preparations containing α2,6-sialic acid [slopes, 1.064 (0.057) and 0.953 (0.033)], particularly in the absence of nonfucosylated forms [0.985 (0.044)].

Conclusions: Glyco-engineered recTSH preparations enriched in sialic acid and inner fucose are promising candidates for future reference materials. These preparations may have advantages over existing preparations used for standardizing TSH measurements.
Evaluation of the Evidence for Rehabilitation Following Acquired Brain Injury
Authors: Turner-Stokes Lynne
Published: November 2005

This article represents a summary of the George Burniston Oration of the Australian Faculty of Rehabilitation Medicine, which was given as a keynote address at the 6th World Congress on Brain Injury, Melbourne, May 2005. The paper concerns evaluation of the evidence for effectiveness of rehabilitation following acquired brain injury (ABI). It explores the current evidence available, and discusses a number of different review strategies which may be used to overcome the challenges of assimilating research evidence in this context.
Behavioural and Psychiatric Symptoms in Patients With Neurodegenerative Diseases
Authors: Lomax Claire , Brown Richard G. , Chaudhuri K. Ray , Howard Robert J.
Published: November 2005

Neuropsychiatric symptoms are common features in patients with neurodegenerative diseases, and have important clinical implications for both patients and caregivers. This study used the Neuropsychiatric Inventory (NPI) to measure symptomatology in 120 patients drawn from five disease groups: Parkinson's disease (PD), Alzheimer's disease (AD), dementia associated with Lewy bodies (DLB), multiple system atrophy (MSA) and progressive supranuclear palsy (PSP). Ninety per cent of the patient group reported experiencing at least one symptom, with the most prevalent symptoms being depression and apathy. However, a differential pattern of symptomatology was reported across the disease groups, a finding which has diagnostic and treatment implications. Furthermore, this provides additional evidence that a broad cortical–subcortical distinction cannot be supported.
Cognitive Impairment in Parkinson's Disease: Is It a Unified Phenomenon?
Authors: Lowit Anja , Howell Peter , Brendel Bettina
Published: November 2005

Parkinson's disease (PD) has long been associated with dementia. This has been found to correlate with participant age, age at onset of PD and severity of PD. In addition, a large corpus of research points to the fact that participants with, as well as without, dementia can be impaired in a variety of cognitive tasks. Among these, set-shifting and dual-tasking skills have received particular focus. Most studies report that a reduction in attentional resources can lead to problems with these tasks. However, none have been able to determine exactly which systems are involved in these skills and which neurological impairments underlie the observed cognitive deficits. The current study set out to investigate how performance on tasks requiring set-shifting and dual tasking related to each other, as well as overall measures of cognition gained across a variety of tasks. Fifteen participants with PD and 12 control participants underwent screening tests for dementia, as well as specific tests to assess attention, set-shifting and dual tasking. The results indicate that set-shifting ability correlated well with other measures of cognitive performance, whereas dualtasking skills did not. This could suggest that set-shifting and dual tasking are not necessarily controlled by the same process, or that a particular process is involved to different degrees. In addition, many participants showed individual performance variations and dissociations between tasks that were not necessarily evident from the statistical analysis. This indicates that it can be difficult to make assumptions on overall cognitive performance from specific tasks and vice versa. This observation has implications for clinical practice as well as research methodology.
Adjusting to Change Through Song: Themes in Songs Written by Clients With Traumatic Brain Injury
Authors: Baker Felicity , Kennelly Jeanette , Tamplin Jeanette
Published: November 2005

The study aimed to identify themes in songs written by clients with traumatic brain injury (TBI). Lyrics (1834) from 82 songs written by 11 female and 21 male clients aged between 5 and 60 years were categorised into 8 main themes and 24 subcategories. Incidence of subcategories and themes were calculated. Self-reflections and messages were the most frequent themes portrayed within songs. Memories and reflections about significant others were also frequently communicated. It was noticeable that clients felt safer to communicate thoughts and feelings about the past and present but were less inclined to confront the future.
Rational engineering of a fluorescein-binding anticalin for improved ligand affinity
Authors: Vopel Sven , Mühlbach Hermine , Skerra Arne
Published: November 2005

The anticalin FluA is an artificial lipocalin with novelspecificity for the fluorescein group, which was engineered from an insect bilin-binding protein by targeted random mutagenesis and selection. Based on the crystal structure of FluA, an attempt was made to improve the complementarity of its ligand pocket to fluorescein by rational protein design. Several side chains participating in sub-optimal interactions with the ligand were identified and replaced by residues that promised a better steric fit. As a result, the substitution of Ala45 by Ile and of Ser114 by Thr or Arg led to a tight affinity of ca. 1 nM, which is approximately 30-fold better than that of the parental anticalin. Similar to the original FluA, the improved version shows almost complete quenching of the bound ligand fluorescence. Interestingly, the quenching effect was significantly reduced when Trp129 was replaced by Tyr, thus supporting the previously postulated role of this residue, which closely packs against the bound ligand, for efficient electron transfer to the excited fluorescein. Circular dichroism spectra revealed that all variants investigated had retained the lipocalin fold. Corresponding thermal unfolding experiments confirmed similar folding stabilities, with melting temperatures ranging from 52.9 to 60.5°C (i.e., for the high-affinity variant).