bluuebunny/crossref_metadata_2025 huggingface dataset:
Doxycycline-regulated gene expression in the opportunistic fungal pathogen Aspergillus fumigatus
Authors: Vogt Keith , Bhabhra Ruchi , Rhodes Judith C , Askew David S
Published: January 2005

Background

Although Aspergillus fumigatus is an important human fungal pathogen there are few expression systems available to study the contribution of specific genes to the growth and virulence of this opportunistic mould. Regulatable promoter systems based upon prokaryotic regulatory elements in the E. coli tetracycline-resistance operon have been successfully used to manipulate gene expression in several organisms, including mice, flies, plants, and yeast. However, the system has not yet been adapted for Aspergillus spp.

Results

Here we describe the construction of plasmid vectors that can be used to regulate gene expression in A. fumigatus using a simple co-transfection approach. Vectors were generated in which the tetracycline transactivator (tTA) or the reverse tetracycline transactivator (rtTA2s-M2) are controlled by the A. nidulans gpdA promoter. Dominant selectable cassettes were introduced into each plasmid, allowing for selection following gene transfer into A. fumigatus by incorporating phleomycin or hygromycin into the medium. To model an essential gene under tetracycline regulation, the E. coli hygromycin resistance gene, hph, was placed under the control of seven copies of the TetR binding site (tetO7) in a plasmid vector and co-transfected into A. fumigatus protoplasts together with one of the two transactivator plasmids. Since the hph gene is essential to A. fumigatus in the presence of hygromycin, resistance to hygromycin was used as a marker of hph reporter gene expression. Transformants were identified in which the expression of tTA conferred hygromycin resistance by activating expression of the tetO7-hph reporter gene, and the addition of doxycycline to the medium suppressed hygromycin resistance in a dose-dependent manner. Similarly, transformants were identified in which expression of rtTA2s-M2 conferred hygromycin resistance only in the presence of doxycycline. The levels of doxycycline required to regulate expression of the tetO7-hph reporter gene were within non-toxic ranges for this organism, and low-iron medium was shown to reduce the amount of doxycycline required to accomplish regulation.

Conclusions

The vectors described in this report provide a new set of options to experimentally manipulate the level of specific gene products in A. fumigatus
General practitioners' reasoning when considering the diagnosis heart failure: a think-aloud study
Authors: Skånér Ylva , Backlund Lars , Montgomery Henry , Bring Johan , Strender Lars-Erik
Published: January 2005

Background

Diagnosing chronic heart failure is difficult, especially in mild cases or early in the course of the disease, and guidelines are not easily implemented in everyday practice. The aim of this study was to investigate general practitioners' diagnostic reasoning about patients with suspected chronic heart failure in comparison with recommendations in European guidelines.

Methods

Think-aloud technique was used. Fifteen general practitioners reasoned about six case vignettes, representing authentic patients with suspected chronic heart failure. Information about each case was added successively in five steps. The general practitioners said their thoughts aloud while reasoning about the probability of the patient having chronic heart failure, and tried to decide about the diagnosis. Arguments for and against chronic heart failure were analysed and compared to recommendations in guidelines.

Results

Information about ejection fraction was the most frequent diagnostic argument, followed by information about cardiac enlargement or pulmonary congestion on chest X-ray. However, in a third of the judgement situations, no information about echocardiography was utilized in the general practitioners' diagnostic reasoning. Only three of the 15 doctors used information about a normal electrocardiography as an argument against chronic heart failure. Information about other cardio-vascular diseases was frequently used as a diagnostic argument.

Conclusions

The clinical information was not utilized to the extent recommended in guidelines. Some implications of our study are that 1) general practitioners need more information about how to utilize echocardiography when diagnosing chronic heart failure, 2) guidelines ought to give more importance to information about other cardio-vascular diseases in the diagnostic reasoning, and 3) guidelines ought to treat the topic of diastolic heart failure in a clearer way.
Lymphocyte subsets in hemophilic patients with hepatitis C virus infection with or without human immunodeficiency virus co-infection: a nested cross-sectional study
Authors: Zhang Mingdong , O'Brien Thomas R , Kopp William C , Goedert James J ,   for the Multicenter Hemophilia Cohort Study
Published: January 2005

Background

With chronic infection, hepatitis C virus (HCV) RNA can be detected in B cells and associated with B-cell disorders, but these are not well defined.

Methods

The relationship between HCV infection and lymphocyte subpopulations was evaluated rigorously in 120 asymptomatic hemophilic patients, randomly selected from a prospective cohort study. CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD56+ NK cells were quantified by flow cytometry using cryopreserved peripheral blood mononuclear cells of 24 hemophilic patients in each of five age-matched groups [uninfected; chronic HCV with or without human immunodeficiency virus (HIV); and cleared HCV with or without HIV].

Results

As expected, patients with HIV had significantly reduced CD4+ and increased CD8+ T cells. Irrespective of HIV, patients with chronic HCV infection had approximately 25% fewer CD19+ B cells than those without chronic HCV infection.

Conclusions

These data support the hypothesis that asymptomatic patients with chronic HCV infection have an altered B-lymphocyte population.
Irradiation specifically sensitises solid tumour cell lines to TRAIL mediated apoptosis
Authors: Marini Patrizia , Schmid Angelika , Jendrossek Verena , Faltin Heidrun , Daniel Peter T , Budach Wilfried , Belka Claus
Published: January 2005

Background

TRAIL (tumor necrosis factor related apoptosis inducing ligand) is an apoptosis inducing ligand with high specificity for malignant cell systems. Combined treatment modalities using TRAIL and cytotoxic drugs revealed highly additive effects in different tumour cell lines. Little is known about the efficacy and underlying mechanistic effects of a combined therapy using TRAIL and ionising radiation in solid tumour cell systems. Additionally, little is known about the effect of TRAIL combined with radiation on normal tissues.

Methods

Tumour cell systems derived from breast- (MDA MB231), lung- (NCI H460) colorectal- (Colo 205, HCT-15) and head and neck cancer (FaDu, SCC-4) were treated with a combination of TRAIL and irradiation using two different time schedules. Normal tissue cultures from breast, prostate, renal and bronchial epithelia, small muscle cells, endothelial cells, hepatocytes and fibroblasts were tested accordingly. Apoptosis was determined by fluorescence microscopy and western blot determination of PARP processing. Upregulation of death receptors was quantified by flow cytometry.

Results

The combined treatment of TRAIL with irradiation strongly increased apoptosis induction in all treated tumour cell lines compared to treatment with TRAIL or irradiation alone. The synergistic effect was most prominent after sequential application of TRAIL after irradiation. Upregulation of TRAIL receptor DR5 after irradiation was observed in four of six tumour cell lines but did not correlate to tumour cell sensitisation to TRAIL. TRAIL did not show toxicity in normal tissue cell systems. In addition, pre-irradiation did not sensitise all nine tested human normal tissue cell cultures to TRAIL.

Conclusions

Based on the in vitro data, TRAIL represents a very promising candidate for combination with radiotherapy. Sequential application of ionising radiation followed by TRAIL is associated with an synergistic induction of cell death in a large panel of solid tumour cell lines. However, TRAIL receptor upregulation may not be the sole mechanism by which sensitation to TRAIL after irradiation is induced.
Randomized clinical trial of surgery versus conservative therapy for carpal tunnel syndrome [ISRCTN84286481]
Authors: Martin Brook I , Levenson Linda M , Hollingworth William , Kliot Michel , Heagerty Patrick J , Turner Judith A , Jarvik Jeffrey G
Published: January 2005

Background

Conservative treatment remains the standard of care for treating mild to moderate carpal tunnel syndrome despite a small number of well-controlled studies and limited objective evidence to support current treatment options. There is an increasing interest in the usefulness of wrist magnetic resonance imaging could play in predicting who will benefit for various treatments.

Method and design

Two hundred patients with mild to moderate symptoms will be recruited over 3 1/2 years from neurological surgery, primary care, electrodiagnostic clinics. We will exclude patients with clinical or electrodiagnostic evidence of denervation or thenar muscle atrophy.

We will randomly assign patients to either a well-defined conservative care protocol or surgery. The conservative care treatment will include visits with a hand therapist, exercises, a self-care booklet, work modification/ activity restriction, B6 therapy, ultrasound and possible steroid injections. The surgical care would be left up to the surgeon (endoscopic vs. open) with usual and customary follow-up. All patients will receive a wrist MRI at baseline.

Patients will be contacted at 3, 6, 9 and 12 months after randomization to complete the Carpal Tunnel Syndrome Assessment Questionnaire (CTSAQ). In addition, we will compare disability (activity and work days lost) and general well being as measured by the SF-36 version II. We will control for demographics and use psychological measures (SCL-90 somatization and depression scales) as well as EDS and MRI predictors of outcomes.

Discussion

We have designed a randomized controlled trial which will assess the effectiveness of surgery for patients with mild to moderate carpal tunnel syndrome. An important secondary goal is to study the ability of MRI to predict patient outcomes.
A primary care, multi-disciplinary disease management program for opioid-treated patients with chronic non-cancer pain and a high burden of psychiatric comorbidity
Authors: Chelminski Paul R , Ives Timothy J , Felix Katherine M , Prakken Steven D , Miller Thomas M , Perhac J Stephen , Malone Robert M , Bryant Mary E , DeWalt Darren A , Pignone Michael P
Published: January 2005

Background

Chronic non-cancer pain is a common problem that is often accompanied by psychiatric comorbidity and disability. The effectiveness of a multi-disciplinary pain management program was tested in a 3 month before and after trial.

Methods

Providers in an academic general medicine clinic referred patients with chronic non-cancer pain for participation in a program that combined the skills of internists, clinical pharmacists, and a psychiatrist. Patients were either receiving opioids or being considered for opioid therapy. The intervention consisted of structured clinical assessments, monthly follow-up, pain contracts, medication titration, and psychiatric consultation. Pain, mood, and function were assessed at baseline and 3 months using the Brief Pain Inventory (BPI), the Center for Epidemiological Studies-Depression Scale scale (CESD) and the Pain Disability Index (PDI). Patients were monitored for substance misuse.

Results

Eighty-five patients were enrolled. Mean age was 51 years, 60% were male, 78% were Caucasian, and 93% were receiving opioids. Baseline average pain was 6.5 on an 11 point scale. The average CESD score was 24.0, and the mean PDI score was 47.0. Sixty-three patients (73%) completed 3 month follow-up. Fifteen withdrew from the program after identification of substance misuse. Among those completing 3 month follow-up, the average pain score improved to 5.5 (p = 0.003). The mean PDI score improved to 39.3 (p < 0.001). Mean CESD score was reduced to 18.0 (p < 0.001), and the proportion of depressed patients fell from 79% to 54% (p = 0.003). Substance misuse was identified in 27 patients (32%).

Conclusions

A primary care disease management program improved pain, depression, and disability scores over three months in a cohort of opioid-treated patients with chronic non-cancer pain. Substance misuse and depression were common, and many patients who had substance misuse identified left the program when they were no longer prescribed opioids. Effective care of patients with chronic pain should include rigorous assessment and treatment of these comorbid disorders and intensive efforts to insure follow up.
Patient satisfaction with out-of-hours primary care in the Netherlands
Authors: van Uden CJT , Ament AJHA , Hobma SO , Zwietering PJ , Crebolder HFJM
Published: January 2005

Background

In recent years out-of-hours primary care in the Netherlands has changed from practice-based to large-scale cooperatives. The purpose of this study is to determine patient satisfaction with current out-of-hours care organised in general practitioner (GP) cooperatives, and gain insight in factors associated with this satisfaction.

Methods

From March to June 2003, 2805 questionnaires were sent to patients within three weeks after they had contacted the GP cooperative in their region. The study was conducted in the province of Limburg in the South of the Netherlands. One-third of these questionnaires was sent to patients who had only received telephone advice, one-third to patients who attended the GP cooperative for consultation, and one-third to patients who received a home visit. Four weeks after the first reminder, a non-respondents telephone interview was performed among a random sample of 100 patients. Analyses were performed with respect to the type of consultation.

Results

The total response was 42.4% (1160/2733). Sixty-seven percent of patients who received telephone advice only reported to be satisfied with out-of-hours care. About 80% of patients who went to the GP cooperative for consultation or those receiving a home visit, reported to be satisfied. Factors that were strongly associated with overall satisfaction included, the doctor's assistant's attitude on the phone, opinion on GP's treatment, and waiting time.

Conclusion

Patients seem generally satisfied with out-of-hours primary care as organised in GP cooperatives. However, patients who received telephone advice only are less satisfied compared to those who attended the GP cooperative or those who received a home visit.
Characterization and performance of a toluene-degrading biofilm developed on pumice stones
Authors: Di Lorenzo Alessandra , Varcamonti Mario , Parascandola Palma , Vignola Rodolfo , Bernardi Adriano , Sacceddu Pasquale , Sisto Raffaello , de Alteriis Elisabetta
Published: January 2005

Background

Hydrocarbon-degrading biofilms in the treatment of contaminated groundwaters have received increasing attention due to the role played in the so-called "biobarriers". These are bioremediation systems in which a microbial consortium adherent to a solid support is placed across the flow of a contaminated plume, thus promoting biodegradation of the pollutant.

Results

A microbial consortium adherent to pumice granules (biofilm) developed from a toluene-enriched microflora in a mini-scale system, following continuous supply of a mineral medium containing toluene, over a 12-month period. Observation by scanning electron microscopy, together with quantification of the biomass attached to pumice, evidenced the presence of abundant exopolymeric material surrounding the cells in the biofilm. Toluene removal monitored during 12-month operation, reached 99%. Identification of the species, based on comparative 16S ribosomal DNA (rDNA) sequence analysis, revealed that Rhodococcus erythropolis and Pseudomonas marginalis were the predominant bacterial species in the microbial consortium.

Conclusion

A structurally complex toluene-degrading biofilm, mainly formed by Rhodococcus erythropolis and Pseudomonas marginalis, developed on pumice granules, in a mini-scale apparatus continuously fed with toluene.
Clinical and echocardiographic features of aorto-atrial fistulas
Authors: Ananthasubramaniam Karthik
Published: January 2005

Aorto-atrial fistulas (AAF) are rare but important pathophysiologic conditions of the aorta and have varied presentations such as acute pulmonary edema, chronic heart failure and incidental detection of the fistula. A variety of mechanisms such as aortic dissection, endocarditis with pseudoaneurysm formation, post surgical scenarios or trauma may precipitate the fistula formation. With increasing survival of patients, particularly following complex aortic reconstructive surgeries and redo valve surgeries, recognition of this complication, its clinical features and echocardiographic diagnosis is important. Since physical exam in this condition may be misleading, echocardiography serves as the cornerstone for diagnosis. The case below illustrates aorto-left atrial fistula formation following redo aortic valve surgery with slowly progressive symptoms of heart failure. A brief review of the existing literature of this entity is presented including emphasis on echocardiographic diagnosis and treatment.
Sister Joseph's nodule in a liver transplant recipient: Case report and mini-review of literature
Authors: Panaro Fabrizio , Andorno Enzo , Di Domenico Stefano , Morelli Nicola , Bottino Giuliano , Mondello Rosalia , Miggino Marco , Jarzembowski Tomasz M , Ravazzoni Ferruccio , Casaccia Marco , Valente Umberto
Published: January 2005

Background

Umbilical metastasis is one of the main characteristic signs of extensive neoplastic disease and is universally referred to as Sister Mary Joseph's nodule.

Case presentation

A 59-years-old Caucasian female underwent liver transplant for end stage liver disease due to hepatitis C with whole graft from cadaveric donor in 2003. After transplantation the patient developed multiple subcutaneous nodules in the umbilical region and bilateral inguinal lymphadenopathy. The excision biopsy of the umbilical mass showed the features of a poorly differentiated papillary serous cystadenocarcinoma. Computed tomographic scan and transvaginal ultrasonography were unable to demonstrate any primary lesion. Chemotherapy was start and the dosage of the immunosuppressive drugs was reduced. To date the patient is doing well and liver function is normal.

Conclusions

The umbilical metastasis can arise from many sites. In some cases, primary tumor may be not identified; nonetheless chemotherapy must be administrated based on patient's history, anatomical and histological findings.
Prognostic significance of metallothionein expression in renal cell carcinoma
Authors: Mitropoulos Dionisios , Kyroudi-Voulgari Aspasia , Theocharis Stamatis , Serafetinides Efraim , Moraitis Epaminondas , Zervas Anastasios , Kittas Christos
Published: January 2005

Background

Metallothionein (MT) protein expression deficiency has been implicated in carcinogenesis while MT over expression in tumors is indicative of tumor resistance to anti-cancer treatment. The purpose of the study was to examine the expression of MT expression in human renal cell carcinoma (RCC) and to correlate MT positivity, the pattern and extent of MT expression with tumor histologic cell type and nuclear grade, pathologic stage and patients' survival.

Patients and methods

The immunohistochemical expression of MT was determined in 43 formalin-fixed and paraffin-embedded RCC specimens, using a mouse monoclonal antibody that reacts with both human MT-I and MT-II. Correlation was sought between immunohistochemical (MT positivity, intensity and extension of staining) and clinico-pathological data (histological cell type, tumor nuclear grade, pathologic stage and patients' survival).

Results

Positive MT staining was present in 21 cases (49%), being mild/moderate and intense in 8 and 13 cases, respectively. The pattern was cytoplasmic in 7 cases and was both cytoplasmic and nuclear in 14 cases. MT expression in a percentage of up to 25% of tumor cells (negative MT staining included) was observed in 31 cases, in a percentage 25–50% of tumor cells in 7 cases, and in a percentage of 50–75% of tumor cells in 5 cases. There was no significant correlation of MT intensity of staining to histological type, stage and patients' survival, while it was inversely correlated to higher tumor nuclear grade. MT extent of staining did not correlate with histological type, nuclear grade, and pathologic stage while a statistically significant association was found with patients' survival.

Conclusions

The inverse correlation between MT staining intensity and tumor nuclear grade in RCC suggests a role of MT in tumor differentiation process. Since extent of MT expression is inversely correlated with survival it may be possibly used as a clinical prognostic parameter.
Evidence for preferential copackaging of Moloney murine leukemia virus genomic RNAs transcribed in the same chromosomal site
Authors: Kharytonchyk Sergey A , Kireyeva Alla I , Osipovich Anna B , Fomin Igor K
Published: January 2005

Background

Retroviruses have a diploid genome and recombine at high frequency. Recombinant proviruses can be generated when two genetically different RNA genomes are packaged into the same retroviral particle. It was shown in several studies that recombinant proviruses could be generated in each round of HIV-1 replication, whereas the recombination rates of SNV and Mo-MuLV are 5 to 10-fold lower. The reason for these differences is not clear. One possibility is that these retroviruses may differ in their ability to copackage genomic RNAs produced at different chromosomal loci.

Results

To investigate whether there is a difference in the efficiency of heterodimer formation when two proviruses have the same or different chromosomal locations, we introduced two different Mo-MuLV-based retroviral vectors into the packaging cell line using either the cotransfection or sequential transfection procedure. The comparative study has shown that the frequency of recombination increased about four-fold when the cotransfection procedure was used. This difference was not associated with possible recombination of retroviral vectors during or after cotransfection and the ratios of retroviral virion RNAs were the same for two variants of transfection.

Conclusions

The results of this study indicate that a mechanism exists to enable the preferential copackaging of Mo-MuLV genomic RNA molecules that are transcribed on the same DNA template. The properties of Mo-MuLV genomic RNAs transport, processing or dimerization might be responsible for this preference. The data presented in this report can be useful when designing methods to study different aspects of replication and recombination of a diploid retroviral genome.
Inflammatory peptides derived from adipose tissue
Authors: Rudin Eric , Barzilai Nir
Published: January 2005

The low-grade inflammation seen with aging is noted particularly in subjects with the metabolic syndrome of aging. Insulin resistance, obesity/abdominal obesity, and risks for many age-related diseases characterize this common syndrome. It is becoming clear that this increased adipose tissue is not simply a reservoir for excess nutrients, but rather an active and dynamic organ capable of expressing several cytokines and other fat-derived peptides (FDP). Some, but not all, FDP may have a role in development of the metabolic syndrome but there is no evidence that these FDP are causing inflammation directly. We suggest that high levels of inflammatory peptides are markers for obesity/abdominal obesity seen with aging, but some may not necessarily have a causative role in the development of inflammation.
Linking biogeography to physiology: Evolutionary and acclimatory adjustments of thermal limits
Authors: Somero George N
Published: January 2005

Temperature-adaptive physiological variation plays important roles in latitudinal biogeographic patterning and in setting vertical distributions along subtidal-to-intertidal gradients in coastal marine ecosystems. Comparisons of congeneric marine invertebrates reveal that the most warm-adapted species may live closer to their thermal tolerance limits and have lower abilities to increase heat tolerance through acclimation than more cold-adapted species. In crabs and snails, heart function may be of critical importance in establishing thermal tolerance limits. Temperature-mediated shifts in gene expression may be critical in thermal acclimation. Transcriptional changes, monitored using cDNA microarrays, have been shown to differ between steady-state thermal acclimation and diurnal temperature cycling in a eurythermal teleost fish (Austrofundulus limnaeus). In stenothermal Antarctic notothenioid fish, losses in capacity for temperature-mediated gene expression, including the absence of a heat-shock response, may reduce the abilities of these species to acclimate to increased temperatures. Differences among species in thermal tolerance limits and in the capacities to adjust these limits may determine how organisms are affected by climate change.
An evaluation of the metabolic syndrome in the HyperGEN study
Authors: Kraja Aldi T , Hunt Steven C , Pankow James S , Myers Richard H , Heiss Gerardo , Lewis Cora E , Rao DC , Province Michael A
Published: January 2005

Background

In 2001 the National Cholesterol Education Program (NCEP) provided a categorical definition for metabolic syndrome (c-MetS). We studied the extent to which two ethnic groups, Blacks and Whites were affected by c-MetS. The groups were members of the Hypertension Genetic Epidemiology Network (HyperGEN), a part of the Family Blood Pressure Program, supported by the NHLBI. Although the c-MetS definition is of special interest in particular to the clinicians, the quantitative latent traits of the metabolic syndrome (MetS) are also important in order to gain further understanding of its etiology. In this study, quantitative evaluation of the MetS latent traits (q-MetS) was based on the statistical multivariate method factor analysis (FA).

Results

The prevalence of the c-MetS was 34% in Blacks and 39% in Whites. c-MetS showed predominance of obesity, hypertension, and dyslipidemia. Three and four factor domains were identified through FA, classified as "Obesity," "Blood pressure," "Lipids," and "Central obesity." They explained approximately 60% of the variance in the 11 original variables. Two factors classified as "Obesity" and "Central Obesity" overlapped when FA was performed without rotation. All four factors in FA with Varimax rotation were consistent between Blacks and Whites, between genders and also after excluding type 2 diabetes (T2D) participants. Fasting insulin (INS) associated mainly with obesity and lipids factors.

Conclusions

MetS in the HyperGEN study has a compound phenotype with separate domains for obesity, blood pressure, and lipids. Obesity and its relationship to lipids and insulin is clearly the dominant factor in MetS. Linkage analysis on factor scores for components of MetS, in familial studies such as HyperGEN, can assist in understanding the genetic pathways for MetS and their interactions with the environment, as a first step in identifying the underlying pathophysiological causes of this syndrome.
Molecular Pain, a New Era of Pain Research and Medicine
Authors: Gu Jianguo , Zhuo Min , Caterina Michael , MacDermott Amy B , Malmberg Annika , Neugebauer Volker , Yoshimura Megumu
Published: January 2005

Molecular pain is a relatively new and rapidly expanding research field that represents an advanced step from conventional pain research. Molecular pain research addresses physiological and pathological pain at the cellular, subcellular and molecular levels. These studies integrate pain research with molecular biology, genomics, proteomics, modern electrophysiology and neurobiology. The field of molecular pain research has been rapidly expanding in the recent years, and has great promise for the identification of highly specific and effective targets for the treatment of intractable pain. Although several existing journals publish articles on classical pain research, none are specifically dedicated to molecular pain research. Therefore, a new journal focused on molecular pain research is needed. Molecular Pain, an Open Access, peer-reviewed, online journal, will provide a forum for molecular pain scientists to communicate their research findings in a targeted manner to others in this important and growing field.
Propofol Suppresses Synaptic Responsiveness of Somatosensory Relay Neurons to Excitatory Input by Potentiating GABAA Receptor Chloride Channels
Authors: Ying Shui-Wang , Goldstein Peter A
Published: January 2005

Propofol is a widely used intravenous general anesthetic. Propofol-induced unconsciousness in humans is associated with inhibition of thalamic activity evoked by somatosensory stimuli. However, the cellular mechanisms underlying the effects of propofol in thalamic circuits are largely unknown. We investigated the influence of propofol on synaptic responsiveness of thalamocortical relay neurons in the ventrobasal complex (VB) to excitatory input in mouse brain slices, using both current- and voltage-clamp recording techniques. Excitatory responses including EPSP temporal summation and action potential firing were evoked in VB neurons by electrical stimulation of corticothalamic fibers or pharmacological activation of glutamate receptors. Propofol (0.6 – 3 μM) suppressed temporal summation and spike firing in a concentration-dependent manner. The thalamocortical suppression was accompanied by a marked decrease in both EPSP amplitude and input resistance, indicating that a shunting mechanism was involved. The propofol-mediated thalamocortical suppression could be blocked by a GABAA receptor antagonist or chloride channel blocker, suggesting that postsynaptic GABAA receptors in VB neurons were involved in the shunting inhibition. GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) were evoked in VB neurons by electrical stimulation of the reticular thalamic nucleus. Propofol markedly increased amplitude, decay time, and charge transfer of GABAA IPSCs. The results demonstrated that shunting inhibition of thalamic somatosensory relay neurons by propofol at clinically relevant concentrations is primarily mediated through the potentiation of the GABAA receptor chloride channel-mediated conductance, and such inhibition may contribute to the impaired thalamic responses to sensory stimuli seen during propofol-induced anesthesia.
Sensitization of TRPV1 by EP1 and IP Reveals Peripheral Nociceptive Mechanism of Prostaglandins
Authors: Moriyama Tomoko , Higashi Tomohiro , Togashi Kazuya , Iida Tohko , Segi Eri , Sugimoto Yukihiko , Tominaga Tomoko , Narumiya Shuh , Tominaga Makoto
Published: January 2005

Prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2) are major inflammatory mediators that play important roles in pain sensation and hyperalgesia. The role of their receptors (EP and IP, respectively) in inflammation has been well documented, although the EP receptor subtypes involved in this process and the underlying cellular mechanisms remain to be elucidated. The capsaicin receptor TRPV1 is a nonselective cation channel expressed in sensory neurons and activated by various noxious stimuli. TRPV1 has been reported to be critical for inflammatory pain mediated through PKA- and PKC-dependent pathways. PGE2 or PGI2increased or sensitized TRPV1 responses through EP1 or IP receptors, respectively predominantly in a PKC-dependent manner in both HEK293 cells expressing TRPV1 and mouse DRG neurons. In the presence of PGE2 or PGI2, the temperature threshold for TRPV1 activation was reduced below 35°C, so that temperatures near body temperature are sufficient to activate TRPV1. A PKA-dependent pathway was also involved in the potentiation of TRPV1 through EP4 and IP receptors upon exposure to PGE2 and PGI2, respectively. Both PGE2-induced thermal hyperalgesia and inflammatory nociceptive responses were diminished in TRPV1-deficient mice and EP1-deficient mice. IP receptor involvement was also demonstrated using TRPV1-deficient mice and IP-deficient mice. Thus, the potentiation or sensitization of TRPV1 activity through EP1 or IP activation might be one important mechanism underlying the peripheral nociceptive actions of PGE2 or PGI2.
A P2X Receptor-Mediated Nociceptive Afferent Pathway to Lamina I of the Spinal Cord
Authors: Chen Meng , Gu Jianguo G
Published: January 2005

Of the six lamina regions in the dorsal horn of the spinal cord, lamina I is a major sensory region involved in nociceptive transmission under both physiological and pathological conditions. While P2X receptors have been shown to be involved in nociception, it remains unknown if P2X receptors are involved in nociceptive transmission to lamina I neurons. Using rat spinal cord slice preparations and patch-clamp recordings, we have demonstrated that the excitatory synaptic transmission between primary afferent fibers and lamina I neurons is significantly affected by ATP and α,β-methylene-ATP. The synaptic effects of them include the increases of the frequency of both miniature excitatory postsynaptic currents (mEPSCs) and spontaneous EPSCs (sEPSCs), and decreases of evoked EPSCs (eEPSCs). These effects were blocked by pyridoxalphosphate-6-azophenyl-2′, 4′-disulfonic acid (PPADS, 10 μM) and suramin (30 μM). In the neurons for which ATP and α,β-methylene-ATP had effects on mEPSCs, sEPSCs and eEPSCs, capsaicin produced similar synaptic effects. Our results indicate that P2X receptors are expressed on many afferent fibers that directly synapse to lamina I neurons. Furthermore, these P2X receptor-expressing afferent fibers are capsaicin-sensitive nociceptive afferents. Thus, this study reveals a P2X receptor-mediated nociceptive afferent pathway to lamina I of the spinal cord and provides a new insight into the nociceptive functions of P2X receptors.
Infiltration of the synovial membrane with macrophage subsets and polymorphonuclear cells reflects global disease activity in spondyloarthropathy
Authors: Baeten Dominique , Kruithof Elli , De Rycke Leen , Boots Anemieke M , Mielants Herman , Veys Eric M , De Keyser Filip
Published: January 2005

Considering the relation between synovial inflammation and global disease activity in rheumatoid arthritis (RA) and the distinct but heterogeneous histology of spondyloarthropathy (SpA) synovitis, the present study analyzed whether histopathological features of synovium reflect specific phenotypes and/or global disease activity in SpA. Synovial biopsies obtained from 99 SpA and 86 RA patients with active knee synovitis were analyzed for 15 histological and immunohistochemical markers. Correlations with swollen joint count, serum C-reactive protein concentrations, and erythrocyte sedimentation rate were analyzed using classical and multiparameter statistics. SpA synovitis was characterized by higher vascularity and infiltration with CD163+ macrophages and polymorphonuclear leukocytes (PMNs) and by lower values for lining-layer hyperplasia, lymphoid aggregates, CD1a+ cells, intracellular citrullinated proteins, and MHC–HC gp39 complexes than RA synovitis. Unsupervised clustering of the SpA samples based on synovial features identified two separate clusters that both contained different SpA subtypes but were significantly differentiated by concentration of C-reactive protein and erythrocyte sedimentation rate. Global disease activity in SpA correlated significantly with lining-layer hyperplasia as well as with inflammatory infiltration with macrophages, especially the CD163+ subset, and with PMNs. Accordingly, supervised clustering using these synovial parameters identified a cluster of 20 SpA patients with significantly higher disease activity, and this finding was confirmed in an independent SpA cohort. However, multiparameter models based on synovial histopathology were relatively poor predictors of disease activity in individual patients. In conclusion, these data indicate that inflammatory infiltration of the synovium with CD163+ macrophages and PMNs as well as lining-layer hyperplasia reflect global disease activity in SpA, independently of the SpA subtype. These data support a prominent role for innate immune cells in SpA synovitis and warrant further evaluation of synovial histopathology as a surrogate marker in early-phase therapeutic trials in SpA.
FcγRIIIB stimulation promotes β1 integrin activation in human neutrophils
Authors: Ortiz-Stern Alejandro , Rosales Carlos
Published: January 2005

The molecular stimuli involved in receptor-induced integrin activation are still poorly defined. We have investigated the role of receptors for the Fc portion of immunoglobulin G molecules (FcγR) on activation of integrins in human neutrophils. Cross-linking of FcγRIIA induced an increase in surface expression of β2 integrins but had no effect on β1 integrins. In contrast, cross-linking of FcγRIIIB not only increased β2 integrins on the cell surface but also induced β1 integrin activation, as indicated by an increase in binding to fibronectin and the appearance of an activation epitope detected by the monoclonal antibody 15/7. The FcγRIIIB-induced increase of β2 integrins required Src-family tyrosine kinases, Syk kinase, and phosphatidylinositol-3 kinase (PI-3K), as the corresponding, specific inhibitors, PP2, Piceatannol, and LY294002, completely blocked it. Contrary to this, FcγRIIIB-indued β1 integrin activation was not blocked by PP2 or LY294002. It was, however, enhanced by Piceatannol. After FcγRIIIB cross-linking, colocalization of FcγRIIIB and active β1 integrins was detected on the neutrophil membrane. These data show, for the first time, that cross-linking of FcγRIIIB induces an inside-out signaling pathway that leads to β1 integrin activation. This activation is independent of Src-family kinases, and PI-3K and may be induced in part by the interaction of FcγRIIIB with β1 integrins.
A Molecular Cascade Showing Nitric Oxide-Heme Oxygenase-1-Vascular Endothelial Growth Factor-Interleukin-8 Sequence in Human Endothelial Cells
Authors: Pae Hyun-Ock , Oh Gi-Su , Choi Byung-Min , Kim Young-Myeong , Chung Hun-Taeg
Published: January 2005

Heme oxygenase (HO)-1 has been shown to be an important biological target of nitric oxide (NO). NO can induce HO-1 expression and IL-8 production, particularly, in endothelial cells. Interestingly, HO-1 tends to induce the production of vascular endothelial growth factor (VEGF) that is involved in endothelial IL-8 syntheses. Whether HO-1 expression by NO may provide a link with IL-8 or VEGF synthesis was investigated in human umbilical vein endothelial cells (HUVECs). The NO donor S-nitroso-N-acetyl-penicillamine (SNAP) dose-dependently increased IL-8 and VEGF productions and HO-1 expression in HUVECs. Transfection with either HO-1 small interfering RNA or HO-1 antisense oligodeoxynucleotide abrogated the ability of SNAP to induce HO-1 expression and IL-8 and VEGF productions. Both pharmacological induction and gene transfer of HO-1 directly induced IL-8 and VEGF productions. Anti-VEGF neutralizing antibody blocked SNAP-mediated IL-8 production and VEGF itself induced IL-8 production, whereas anti-IL-8 neutralizing antibody had no effect on VEGF production in SNAP-treated HUVECs. Neither anti-VEGF nor anti-IL-8 antibodies influenced SNAP-induced HO-1 expression. Moreover, neither VEGF nor IL-8 showed an additive effect on SNAP-induced HO-1 expression. HO-1 transfection had no significant effect on productions of other CXC chemokines, such as growth-related oncogen-α and epithelial neutrophil activation peptide-78. Taken together, these results provide a molecular cascade showing NO-HO-1-VEGF-IL-8 sequence in human endothelial cells.
Coactivator Proteins as Determinants of Estrogen Receptor Structure and Function: Spectroscopic Evidence for a Novel Coactivator-Stabilized Receptor Conformation
Authors: Tamrazi Anobel , Carlson Kathryn E. , Rodriguez Alice L. , Katzenellenbogen John A.
Published: January 2005

The direct regulation of gene transcription by nuclear receptors, such as the estrogen receptor (ER), involves not just ligand and DNA binding but the recruitment of coregulators. Typically, recruitment of p160 coactivator proteins to agonist-liganded ER is considered to be unidirectional, with ligand binding stabilizing an ER ligand binding domain (LBD) conformation that favors coactivator interaction. Using fluorophore-labeled ERα-LBDs, we present evidence for a pronounced stabilization of ER conformation that results from coactivator binding, manifest by decreased ER sensitivity to proteases and reduced conformational dynamics, as well as for the formation of a novel coactivator-stabilized (costabilized) receptor conformation, that can be conveniently monitored by the generation of an excimer emission from pyrene-labeled ERα-LBDs. This costabilized conformation may embody features required to support ER transcriptional activity. Different classes of coactivator proteins combine with estrogen agonists of different structure to elicit varying degrees of this receptor stabilization, and antagonists and coactivator binding inhibitors disfavor the costabilized conformation. Remarkably, high concentrations of coactivators engender this conformation even in apo- and antagonist-bound ERs (more so with selective ER modulators than with pure antagonists), providing an in vitro model for the development of resistance to hormone therapy in breast cancer.
An evaluation of detection methods for large lariat RNAs
Authors: COOMBES CANDICE E. , BOEKE JEF D.
Published: January 2005

Ty1 elements are long terminal repeat (LTR) retrotransposons that reside within the genome of Saccharomyces cerevisiae. It has been known for many years that the 2′-5′ phosphodiesterase Dbr1p, which debranches intron lariats, is required for efficient Ty1 transposition. A recent report suggested the intriguing possibility that Ty1 RNA forms a lariat as a transposition intermediate. We set out to further investigate the nature of the proposed Ty1 lariat branchpoint. However, using a wide range of techniques we were unable to find any evidence for the proposed lariat structure. Furthermore, we demonstrate that some of the techniques used in the initial study describing the lariat are capable of incorrectly reporting a lariat structure. Thus, the role of the Dbr1 protein in Ty1 retrotransposition remains elusive.
The Historical and Political Background to the Proposals for Local Commissioning of Primary Dental Care by Primary Care Trusts
Authors: Batchelor Paul
Published: January 2005

This paper provides a background to the changes that are about to occur in the system for primary oral healthcare delivery in the NHS. This is part of a far wider reform programme involving all public sector services. The challenges that the Government faces are not unique: all countries are striving to alter their care systems to address the changing expectations of the electorate while attempting to control both the costs and inequalities. What is unique to the United Kingdom is the historical legacy of the NHS and its political importance, which should not be underestimated. An overview of the agenda for change in all parts of the health sector is presented. This is followed by a more detailed analysis of the proposals for future delivery of primary oral care in the NHS and the subsequent issues arising. A consistent theme running throughout the Government's agenda is devolution. Indeed, the detailed programme discussed in this paper applies only to England and Wales. The fact that Scotland has different plans highlights the importance of the political and cultural setting to any reform programme.
Introducing Dentists with Special Interests (DwSIs)— Past, Present and Future Perspectives
Authors: Drake Sharon
Published: January 2005

This paper provides the national context for the development of practitioners with special interests (PwSIs) in light of the recent introduction of the concept to dentistry. Given the shortage of specialists and consultants in some dental specialities and the fact that a number of referrals could be managed in the primary care sector, the development of an additional tier to bridge the gap between current capacity and demand for services in secondary care seems to be a practical solution. The introduction of the DwSI and the future training opportunities it affords will pave the way for the development of a cadre of accredited primary care practitioners with enhanced skills who, together with their secondary care colleagues, will help widen the choice available to patients in terms of the nature and locality of NHS dental care provided. In time, DwSIs may wish to train to become full specialists or consultants and have their accredited prior learning and experience recognised towards completion of full specialist training.
Opportunities for the Dental Team Arising Out of the New Arrangements for Primary Dental Care
Authors: Lambert-Humble Stephen
Published: January 2005

Many changes are occurring in the delivery of dental care to patients as a result of the implementation of a series of Government initiatives to improve the services to patients throughout the National Health Service (NHS). Dentistry is no exception, and the whole dental team should benefit from new opportunities that have opened up as a result of these changes. This paper outlines a number of these initiatives, and describes some of the potential opportunities that may develop for each group within the dental team. Many of these changes are already in the pipeline, many depend on funding, and many depend on a change in the mindset of established organisations and institutions to think ‘outside the box’. The author believes that none of the changes outlined or discussed in this paper challenge the position or status of any group; however, they do require all members of the dental team to examine the way in which they work, to look at the evidence base for these ways of working, and then perhaps look for innovative solutions to the employment, training and development of all members of the team.
Linking Clinical Audit in General Dental Services to Primary Care Trust Clinical Governance—Progress Report of an Approach Used in Southend
Authors: Cannell Phillip
Published: January 2005

Clinical audit has been defined as the systematic, critical analysis of the quality of dental care, including the procedures and processes used for diagnosis, intervention and treatment, the use of resources and the resulting outcome and quality of life as assessed by both professionals and patients.1 The aim of clinical audit is to encourage dentists to self-examine different aspects of their practices, to implement improvements where the need is identified and to re-examine, from time to time, those areas that have been audited to ensure that a high quality of service is being maintained or further improved. Since 1st April 2001, all general dental practitioners (principals and assistants) working in the General Dental Services (GDS) have been required to participate in a rolling programme of at least 15 hours of clinical audit or peer review every three years.2 The first three-year cycle ended on 31st March 2004. By the end of December 2003, 96% of dentists had either undertaken or committed to undertake clinical audit/peer review activities.3 This initiative, in conjunction with the voluntary clinical audit and peer review schemes which preceded it, has provided opportunities for dentists and their practices to use these activities to assist in quality improvements in their practices, for the benefit of their patients. However, there are other methods for carrying out clinical audit and, in the NHS, there is a need to link it to clinical governance. This paper gives a progress report on an approach that has been piloted by Southend Primary Care Trust (PCT). It deals with the rationale for the project and outlines the methods used. It does not report results. These will follow in a subsequent paper.