Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes.

Multiple studies have suggested that the protein kinase Akt/PKB (protein kinase B) is required for insulin-stimulated glucose transport in skeletal muscle and adipose cells. In an attempt to understand links between Akt activation and glucose transport regulation, we applied mass spectrometry-based proteomics and bioinformatics approaches to identify potential Akt substrates containing the phospho-Akt substrate motif RXRXXpS/T. The present study describes the identification of the Rab GAP (GTPase-activating protein)-domain containing protein TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1], which is closely related to TBC1D4 [TBC domain family, member 4, also denoted AS160 (Akt substrate of 160 kDa)], as an Akt substrate that is phosphorylated at Thr(590). RNAi (RNA interference)-mediated silencing of TBC1D1 elevated basal deoxyglucose uptake by approx. 61% in 3T3-L1 mouse embryo adipocytes, while the suppression of TBC1D4 and RapGAP220 under the same conditions had little effect on basal and insulin-stimulated deoxyglucose uptake. Silencing of TBC1D1 strongly increased expression of the GLUT1 glucose transporter but not GLUT4 in cultured adipocytes, whereas the decrease in TBC1D4 had no effect. Remarkably, loss of TBC1D1 in 3T3-L1 adipocytes activated the mTOR (mammalian target of rapamycin)-p70 S6 protein kinase pathway, and the increase in GLUT1 expression in the cells treated with TBC1D1 siRNA (small interfering RNA) was blocked by the mTOR inhibitor rapamycin. Furthermore, overexpression of the mutant TBC1D1-T590A, lacking the putative Akt/PKB phosphorylation site, inhibited insulin stimulation of p70 S6 kinase phosphorylation at Thr(389), a phosphorylation induced by mTOR. Taken together, our data suggest that TBC1D1 may be involved in controlling GLUT1 glucose transporter expression through the mTOR-p70 S6 kinase pathway.

An evaluation of Irish cattle herds with inconclusive serological evidence of bovine brucellosis.

Since 1998, there has been a steady decline in herd restrictions and de-populations in Ireland due to bovine brucellosis. There is concern that the interpretation of laboratory results may become increasingly problematic, as brucellosis prevalence falls in Ireland. Therefore, the purpose of the current study was to evaluate the infection status of Irish herds and animals with inconclusive serological evidence of bovine brucellosis. During 12 months from September 1, 2004, laboratory and observational epidemiological data were collected from all Irish herds where animal testing identified at least one animal with a complement fixation test (CFT) reading greater than zero and/or a positive result to the indirect enzyme-linked immunosorbent assay (iELISA). Due to the observational nature of the study, we have robust estimates of the relative, but not the absolute, performance of the CFT, iELISA and brucellin skin test (BST). Herds were divided into three categories (Group A, B or C) on the basis of test results at initial assessment. A total of 639 herds were enrolled into the study, and observed for at least two years following enrolment. A rising CFT titre, with a CFT reading of 111 International CFT Units (IU) or greater at the subsequent blood test, was generally associated with herds where other evidence of infection was also available. Knowledge of the CFT reading at the initial and a subsequent blood test proved useful in distinguishing false-positive and true-positive brucellosis results. There was poor correlation between the CFT and iELISA results, and between the CFT and BST results. As a result of this study, national policy has been modified to include re-sampling of all animals with CFT readings of 20 IU or greater. This project has also led to a reduction in the number of herds restricted, as well as restriction duration. It has also contributed to a reduction in the number of herds listed for contiguous tests, and therefore the potential for contiguity testing of false positive results.

Superiority of "triple" therapy with salmeterol/fluticasone propionate and tiotropium bromide versus individual components in moderate to severe COPD.

BACKGROUND: The combination of salmeterol and fluticasone propionate (SFC) and tiotropium bromide (TIO) are commonly used treatments in chronic obstructive pulmonary disease (COPD) but there are few data on their effectiveness when used together. We compared the effects of SFC 50/500 microg twice daily in addition to TIO 18 microg once daily with the individual treatments alone. METHODS: 41 patients with COPD participated in a randomised, double blind, double dummy, three way crossover study with 2 week washout periods between treatments. Lung function assessment included plethysmography and spirometry. The primary end point was post-dose specific airways conductance (sGaw) area under the curve (AUC(0-4 h)) on day 14. RESULTS: AUC(0-4 h) sGaw was significantly higher on day 14 after SFC+TIO compared with TIO (22%) or SFC alone (27%) (both p<0.001). SFC+TIO significantly improved trough forced expiratory volume in 1 s compared with TIO alone (212 ml, p<0.001) and SFC alone (110 ml, p = 0.017) on day 14. Inspiratory capacity measurements also showed significant benefits for triple therapy over individual components on day 14. Subjects receiving SFC+TIO had clinically relevant improvements in Transition Dyspnoea Index (TDI) total score of 2.2 compared with TIO alone (p<0.001) (but not SFC alone, 0.7; NS) and used significantly less rescue medication (1.0 occasion less daily than TIO (p<0.001) and 0.6 less than SFC (p = 0.01)). CONCLUSION: SFC+TIO triple therapy led to greater improvements in bronchodilation compared with TIO and SFC alone. The advantages of triple therapy are observed across a range of physiologically important parameters, including airway conductance and lung volumes. Triple therapy also led to patient related benefits by improving TDI and use of rescue medication.

Relationship between lung function impairment and incidence or recurrence of cardiovascular events in a middle-aged cohort.

INTRODUCTION: Lung function impairment may be a risk factor for cardiovascular disease (CVD) events. OBJECTIVE: To determine the relationship between the severity of airflow obstruction based on modified Global Initiative on Obstructive Lung Disease (GOLD) criteria and the prevalence and incidence or recurrence of CVD in a cohort of US adults, aged 45-64 years, from 1987 to 2001. METHODS: We analysed data from 14 681 adults using logistic regression to determine the cross sectional association between lung function impairment and prevalent CVD at baseline and Cox regression to examine the prospective association of lung function impairment at baseline with CVD over 15 years of follow-up. Models were adjusted for age, sex, race, smoking, comorbid hypertension and diabetes, cholesterol levels and fibrinogen level. RESULTS: At baseline, the crude prevalence of CVD was higher among subjects with GOLD 2 (OR 2.9, 95% CI 2.4 to 3.6) and GOLD 3 or 4 chronic obstructive pulmonary disease (COPD) (OR 3.0, 95% CI 2.0 to 4.5), compared with normal subjects. These relative risks were greatly reduced after adjusting for covariates (OR 1.4, 95% CI 1.1 to 1.8 for GOLD 2 and OR 1.3, 95% CI 0.8 to 2.1 for GOLD 3 or 4). Similarly, the association between COPD and risk of incident or recurrent CVD was much stronger in the unadjusted models (hazard ratio (HR) 2.4, 95% CI 2.1 to 2.7 for GOLD 2 and 2.9, 95% CI 2.2 to 3.9 for GOLD 3 or 4) than in the adjusted ones (HR 1.2, 95% CI 1.03 to 1.4 for GOLD 2 and 1.5, 95% CI 1.1 to 2.0 for GOLD 3 or 4). CONCLUSION: We observed a crude association between lung function impairment and prevalent and incident or recurrent CVD that was greatly reduced after adjusting for covariates, including age, sex, race, smoking, comorbid hypertension and diabetes, cholesterol levels and fibrinogen level. These data suggest that this association may be, in part, mediated through established CVD risk factors included in our adjusted models.

Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points (ECLIPSE).

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease and not well understood. The forced expiratory volume in one second is used for the diagnosis and staging of COPD, but there is wide acceptance that it is a crude measure and insensitive to change over shorter periods of time. Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points (ECLIPSE) is a 3-yr longitudinal study with four specific aims: 1) definition of clinically relevant COPD subtypes; 2) identification of parameters that predict disease progression in these subtypes; 3) examination of biomarkers that correlate with COPD subtypes and may predict disease progression; and 4) identification of novel genetic factors and/or biomarkers that both correlate with clinically relevant COPD subtypes and predict disease progression. ECLIPSE plans to recruit 2,180 COPD subjects in Global Initiative for Chronic Obstructive Lung Disease categories II-IV and 343 smoking and 223 nonsmoking control subjects. Study procedures are to be performed at baseline, 3 months, 6 months and every 6 months thereafter. Assessments include pulmonary function measurements (spirometry, impulse oscillometry and plethysmography), chest computed tomography, biomarker measurement (in blood, sputum, urine and exhaled breath condensate), health outcomes, body impedance, resting oxygen saturation and 6-min walking distance. Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points is the largest study attempting to better describe the subtypes of chronic obstructive pulmonary disease, as well as defining predictive markers of its progression.

In vivo assessment of left ventricular remodelling after myocardial infarction by digital video contrast angiography in the rat.

OBJECTIVE: The aim was to develop a digital video contrast angiographic method for assessing global left ventricular function and volume in vivo in the rat and then to apply it to a study of ventricular remodelling after coronary occlusion, with and without reperfusion. METHODS: Digital contrast angiography was performed on 29 rats, including the following groups: sham operated (n = 11), non-transmural myocardial infarction produced by reperfusion (n = 8), and transmural infarction produced by permanent occlusion (n = 10). Under anaesthesia three weeks later, biplane fluoroscopic images were acquired following venous contrast injection. Levophase images were digitised, and left ventricular end diastolic and end systolic volumes and ejection fractions were obtained using an area-length method. Left ventricular ejection fraction data also were calculated by videodensitometry from video density curves. RESULTS: Compared to the sham operated group, the reperfused group showed a significant decrease in left ventricular ejection fraction, at 53(SD 7) v 70(5)% (p < 0.01), and an increase in end diastolic volume. The permanent occlusion group showed a further decrease in the ejection fraction [40(8)%] and a further significant increase in end diastolic volume compared to the reperfused group (p < 0.01). Left ventricular ejection fraction correlated inversely with percent infarct size (r = 0.882) and showed a positive correlation with the spared epicardial area (r = 0.721). Most haemodynamic variables, including maximum left ventricular dP/dt, failed to discriminate between the groups. The methods showed reasonable accuracy when tested in vitro using contrast filled balloons. In vivo, the left ventricular ejection fraction calculated by densitometry showed adequate interobserver variability (2 SD +/- 8.5 percentage points), but the area-length method showed somewhat more scatter. CONCLUSIONS: Digital video contrast angiography is a feasible method for the assessing global left ventricular function in the rat and should be useful in other small animal models. Significant differences in left ventricular volumes and ejection fractions were detected between reperfused and permanent occlusion groups, whereas haemodynamic variables showed non-significant trends. Reperfusion after 45 min of occlusion caused sparing of the epicardium, prevented unfavourable remodelling, and improved the ejection fraction compared to permanent occlusion.

Ultrafast networks (ATM): first clinical experiences.

Ultrafast networks using asynchronous transfer mode (ATM) technology can provide the bandwidth and throughput that may be sufficient to satisfy the medical imaging community. Several trials are underway to assess the effect of ATM network capabilities on the clinical practice of radiology, by providing immediate interactive radiology consultations between subspecialists and general radiologists at affiliated academic institutions. The hardware to build such networks is now commercially available and its cost is decreasing steadily, but the monthly charges for ATM bandwidth use are still high. Nevertheless, given the tremendous increase in communication capability and data transfer rates possible with ATM networks, cost alone should not be the determining factor for selecting this technology. The ATM concept in general is first reviewed, followed by a description of early clinical ATM network installation in four medical environments worldwide. These medical clusters include: the UCLA affiliated hospitals (UCLA Medical Center, West LA VAMC and Olive-View UCLA Medical Center), the UCSF affiliated hospitals, Duke University Hospitals and a cluster of medical centers in Berlin which have all been connected via ATM networks. The use of ATM technology in these realistic clinical environments is discussed and evaluated for its potential impact on patient care and clinical teaching within radiology departments. From this preliminary study it is concluded that image communications over a regional PACS using an ATM network can allow interactive consultations between different subspecialist and general radiologists or other specialized radiologists spread over different medical centers.

A Rictor-Myo1c complex participates in dynamic cortical actin events in 3T3-L1 adipocytes.

Insulin signaling through phosphatidylinositol 3-kinase (PI 3-kinase) activates the protein kinase Akt through phosphorylation of its threonine 308 and serine 473 residues by the PDK1 protein kinase and the Rictor-mammalian target of rapamycin complex (mTORC2), respectively. Remarkably, we show here that the Rictor protein is also present in cultured adipocytes in complexes containing Myo1c, a molecular motor that promotes cortical actin remodeling. Interestingly, the Rictor-Myo1c complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Furthermore, while RNA interference-directed silencing of Rictor results in the expected attenuation of Akt phosphorylation at serine 473, depletion of Myo1c is without effect. In contrast, loss of either Rictor or Myo1c inhibits phosphorylation of the actin filament regulatory protein paxillin at tyrosine 118. Furthermore, Myo1c-induced membrane ruffling of 3T3-L1 adipocytes is also compromised following Rictor knockdown. Interestingly, neither the mTORC2 inhibitor rapamycin nor the PI 3-kinase inhibitor wortmannin affects paxillin tyrosine 118 phosphorylation. Taken together, our findings suggest that the Rictor-Myo1c complex is distinct from mTORC2 and that Myo1c, in conjunction with Rictor, participates in cortical actin remodeling events.

RNAi-based gene silencing in primary mouse and human adipose tissues.

Cultured adipocyte cell lines are a model system widely used to study adipose function, but they exhibit significant physiological differences compared with primary cells from adipose tissue. Here we report short interfering RNA-based methodology to selectively attenuate gene expression in mouse and human primary adipose tissues as a means of rapidly validating findings made in cultured adipocyte cell lines. The method is exemplified by depletion of the PTEN phosphatase in white adipose tissue (WAT) from mouse and humans, which increases Akt phosphorylation as expected. This technology is also shown to silence genes in mouse brown adipose tissue. Previous work revealed upregulation of the mitochondrial protein UCP1 in adipose cells from mice lacking the gene for the transcriptional corepressor RIP140, whereas in cultured adipocytes, loss of RIP140 has a little effect on UCP1 expression. Application of our method to deplete RIP140 in primary mouse WAT elicited markedly increased oxygen consumption and expression of UCP1 that exactly mimics the phenotype observed in RIP140-null mice. This ex-vivo method of gene silencing should be useful in rapid validation studies as well as in addressing the depot- and species-specific functions of genes in adipose biology.

Implementation of British Thoracic Society guidelines for acute exacerbation of chronic obstructive pulmonary disease: impact on quality of life.

BACKGROUND: The British Thoracic Society (BTS) guidelines have not been examined collectively for their impact on chronic obstructive pulmonary disease (COPD). Whether intensive outpatient follow up of COPD patients after acute admission, using these guidelines, improved quality of life compared to the "usual practice" of primary care follow up was investigated. METHODS: Altogether 103 patients with a new diagnosis of COPD were admitted and screened over a four year period. Seventy patients were excluded because of another dominant medical condition or a mandatory requirement for intervention. Patients were randomised to regular primary care (control group, n = 15) or chest clinic follow up (intervention group, n = 10). Spirometry, oxygen saturation, St George's Respiratory Questionnaire (SGRQ), and Short Form 36 questionnaire were measured at baseline and six months. The intervention group was reviewed at least four times in the six month period and received spirometry, ambulatory oxygen assessment, smoking cessation advice, nebuliser assessment, a steroid trial, advice about nutrition/exercise, and introduction to a patient support group. RESULTS: There was no significant difference between baseline measurements in the two groups. There was a significant mean (SD) improvement in the SGRQ symptom score from baseline to six months in the intervention group [20.98 (20.36)] compared with the controls [0.23 (12.55)] (p = 0.004). At six months the SGRQ symptom score, impact score, and total score was significantly better in the intervention than the control group (p = 0.01, 0.02, and 0.02). CONCLUSION: Aggressive implementation of BTS guidelines after initial hospitalisation may improve respiratory health specific quality of life scores in patients with COPD. Larger studies are needed to confirm this finding.

Dosimetric study of microwave cataractogenesis.

A closed waveguide method has been used to induce opacities in the rabbit eye. A 30-min exposure to 2.45-GHz radiation such that 8.7 W is incident on the head (5.75 W being absorbed) produces a cataract in half of the exposed eyes of New Zealand white rabbits.

The role of digital imaging and communications in medicine in an evolving healthcare computing environment: the model is the message.

The decision to use Digital Imaging and Communications in Medicine (DICOM), Health Level 7 (HL7), a common object broker such as the Common Object Request Brokering Architecture (CORBA) or ActiveX (Microsoft Corp, Redmond, WA) or any other protocol for the transfer of DICOM data depends on the requirements of a particular implementation. The selection of protocol is independent of the information model. Our goal as message standards developers is to design a data interchange infrastructure that will faithfully convey the computer-based patient record and make it available to authorized health care providers when and where it is needed for patient care. DICOM accurately and expressively represents the clinically significant properties of images and the semantics of image-related information. The DICOM data model is small and well-defined. The model can be expressed in Standard Generalized Markup Language (SGML) or Object Management Group Interface Definition Language or other common syntax-and can be implemented using any reliable communications protocol. Therefore our opinion is that the DICOM semantic data model should serve as the basis for a logically equivalent set of specifications in HL7, CORBA, ActiveX, and SGML for the interchange of biomedical images and image-related information.

Immunity to type XI collagen in mice. Evidence that the alpha 3(XI) chain of type XI collagen and the alpha 1(II) chain of type II collagen share arthritogenic determinants and induce arthritis in DBA/1 mice.

To determine whether native bovine type XI collagen (BXI) is arthritogenic, five strains of inbred mice were immunized with BXI/CFA. Arthritis was not observed in any of these strains, though it was prevalent in DBA/1 and B10.RIII controls immunized with bovine type II collagen (BII). Antisera from BXI-immunized mice reacted with mouse type XI collagen (MsXI), weakly with the alpha-chains of BXI, and minimally with mouse type II collagen (MsII). However, antisera to BII reacted with MsII and MsXI, indicating antibodies to conformation-independent epitopes shared by alpha 1(II) and alpha 3(XI). Mice immunized with BXI containing a small amount of BII developed arthritis much like those immunized with BII; sera from these mice reacted with MsXI and MsII. Delayed-type hypersensitivity responses differed from IgG responses, i.e., BXI elicited responses to alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II); BII, to alpha 3(XI) and alpha 1(II) exclusively. To determine whether alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II) are arthritogenic, DBA/1J mice were immunized with each alpha-chain. Arthritis was seen in mice injected with alpha 3(XI) or alpha 1(II). Sera to both alpha-chains reacted similarly with MsII and peptide fragment alpha 1(II)-CB11. Epitope mapping using polyclonal and mAb to type II collagen revealed that all polyclonal and 11 of 14 mAb reacted with alpha 3(XI) and alpha 1(II), whereas three mAb reacted only with alpha 1(II). In conclusion, BXI is immunogenic but not arthritogenic in five strains of mice, whereas alpha 3(XI) and alpha 1(II) are arthritogenic and immunogenic in DBA/1 mice and share greater than or equal to 11 epitopes recognized by autoantibody.