The preparation, solubilisation and binding characteristics of a beta 2-adrenoceptor isolated from transfected Chinese hamster cells

beta2-Adrenoceptor agonists (beta -agonists) are well known for their growth promoting and repartitioning effects in many species. Although the use of these compounds to increase muscle mass in stockfarming is prohibited within the EU, under directive 96/22/EC, significant illegal use still occurs. With legal and illegal synthesis of new structurally related compounds, the detection of traditional beta -agonists and new derivatives becomes increasingly problematical. This method describes the isolation and solubilisation of a beta2-adrenoceptor from a transfected Chinese hamster ovary cell line, using the detergent digitonin. The solubilised receptor retained its activity and was isolated from the cell membrane at a concentration of 550 +/- 100 fmol mg(-1) of solubilised protein. Competition analysis using the tritiated antagonist dihydroalprenolol revealed receptor affinity for five structurally different beta -agonists, with IC50 values ranging from 2.1 +/- 0.76 X 10(-7) M for salmeterol to 1.1 +/- 0.62 x 10(-5) M for ractopamine. This study has demonstrated that transfected cell lines with a high expression of beta2-adrenoceptors are a convenient source of active receptor material. Solubilised beta (2)-adrenoceptors could form the basis of a multi-analyte screening assay for use in routine screening.

The preparation, solubilisation and binding characteristics of a beta 2-adrenoceptor isolated from transfected Chinese hamster cells

beta2-Adrenoceptor agonists (beta -agonists) are well known for their growth promoting and repartitioning effects in many species. Although the use of these compounds to increase muscle mass in stockfarming is prohibited within the EU, under directive 96/22/EC, significant illegal use still occurs. With legal and illegal synthesis of new structurally related compounds, the detection of traditional beta -agonists and new derivatives becomes increasingly problematical. This method describes the isolation and solubilisation of a beta2-adrenoceptor from a transfected Chinese hamster ovary cell line, using the detergent digitonin. The solubilised receptor retained its activity and was isolated from the cell membrane at a concentration of 550 +/- 100 fmol mg(-1) of solubilised protein. Competition analysis using the tritiated antagonist dihydroalprenolol revealed receptor affinity for five structurally different beta -agonists, with IC50 values ranging from 2.1 +/- 0.76 X 10(-7) M for salmeterol to 1.1 +/- 0.62 x 10(-5) M for ractopamine. This study has demonstrated that transfected cell lines with a high expression of beta2-adrenoceptors are a convenient source of active receptor material. Solubilised beta (2)-adrenoceptors could form the basis of a multi-analyte screening assay for use in routine screening.

A 76-84 GHz SiGe power amplifier array employing low-loss four-way differential combining transformer

This paper presents holistic design of a novel four-way differential power-combining transformer for use in millimeter-wave power-amplifier (PA). The combiner with an inner radius of 25 µm exhibits a record low insertion loss of 1.25 dB at 83.5 GHz. It is designed to simultaneously act as a balanced-to-unbalanced converter, removing the need for additional BALUNs typically required in differential circuits. A complete circuit comprised of a power splitter, two-stage differential cascode PA array, a power combiner as well as input and output matching elements was designed and realized in SiGe technology with f/f 170/250 GHz. Measured small-signal gain of at least 16.8 dB was obtained from 76.4 to 85.3 GHz with a peak 19.5 dB at 83 GHz. The prototype delivered 12.5 dBm output referred 1 dB compression point and 14 dBm saturated output power when operated from a 3.2 V dc supply voltage at 78 GHz.

HIV-1 Nef limits communication between linker of activated T cells and SLP-76 to reduce formation of SLP-76-signaling microclusters following TCR stimulation

Signal initiation by engagement of the TCR triggers actin rearrangements, receptor clustering, and dynamic organization of signaling complexes to elicit and sustain downstream signaling. Nef, a pathogenicity factor of HIV, disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption, we employed quantitative single-cell microscopy following surface-mediated TCR stimulation that allows for dynamic visualization of distinct signaling complexes as microclusters (MCs). Despite marked inhibition of actin remodeling and cell spreading, the induction of MCs containing TCR-CD3 or ZAP70 was not affected significantly by Nef. However, Nef potently inhibited the subsequent formation of MCs positive for the signaling adaptor Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) to reduce MC density in Nef-expressing and HIV-1-infected T cells. Further analyses suggested that Nef prevents formation of SLP-76 MCs at the level of the upstream adaptor protein, linker of activated T cells (LAT), that couples ZAP70 to SLP-76. Nef did not disrupt pre-existing MCs positive for LAT. However, the presence of the viral protein prevented de novo recruitment of active LAT into MCs due to retargeting of LAT to an intracellular compartment. These modulations in MC formation and composition depended on Nef's ability to simultaneously disrupt both actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus employs a dual mechanism to disturb early TCR signaling by limiting the communication between LAT and SLP-76 and preventing the dynamic formation of SLP-76-signaling MCs.

PTGS2 (Cyclooxygenase-2) expression and survival among colorectal cancer patients: A systematic review

Background: Studies have examined whether tumor expression of PTGS2 (also known as COX-2), an enzyme inhibited by nonsteroidal anti-inflammatory drugs such as aspirin, is associated with prognosis in patients with colorectal cancer. However, results to date have been mixed. Methods: Using terms for PTGS2 and colorectal cancer, the Medline, Embase, and Web of Science databases were systematically searched for studies published, in any language, until December 2011. Random effects meta-analyses were used to calculate pooled HRs [95% confidence intervals (CI)] for the association between PTGS2 expression and tumor recurrence, colorectal cancer-specific survival, and overall survival. Results: In total, 29 studies, which had prognostic data on 5,648 patients, met the inclusion criteria. PTGS2- positive patients were at an increased risk of tumor recurrence (n = 9 studies; HR, 2.79; 95% CI, 1.76-4.41; P <0.001) and had poorer colorectal cancer-specific survival (n = 7; HR, 1.36; 95% CI, 1.02-1.82; P = 0.04). However, there was funnel plot asymmetry, possibly due to publication bias, for the association with cancerspecific survival but less so for recurrence. PTGS2 expression was not associated with overall survival [(n= 16; pooled unadjusted HR, 1.30; 95% CI, 0.94-1.79; P=0.11) and (n=9; pooled adjusted HR, 1.02; 95% CI, 0.72-1.45; P = 0.91)]. Conclusions: PTGS2 expression was associated with an increased risk of tumor recurrence and poorer colorectal cancer-specific survival but not overall survival among patients with colorectal cancer. However, confounding by tumor characteristics such as tumor stage seems likely. Impact: There is insufficient evidence to recommend PTGS2 expression as a prognostic marker in patients with colorectal cancer. Furthermore, studies providing adjusted results are required. © 2013 AACR.

Impact of different health systems on the burden of type 2 diabetes in two populations

Objectives: Health policy directs the management of patients with chronic disease in a country, but evaluating nationwide policies is difficult, not least because of the absence of suitable comparators. This paper examines the management of patients with type 2 diabetes in two demographically comparable populations with different health care systems to see if this represents a viable approach to evaluation.

Methods: A secondary analysis of centralized prescribing databases for 2010 was undertaken to compare the levels and costs of care of patients with type 2 diabetes in Northern Ireland’s National Health Service (NHS) (NI, n = 1.8 million) which has structured care, financial incentives related to diabetes care and an emphasis on generic prescribing, with that of the Republic of Ireland (ROI, n = 4.3 million) where management of diabetes care is guided solely by clinical and other guidelines.

Results: The prevalence of treated type 2 diabetes was 3.59% in NI and 3.09% in ROI, but there were similar and high levels of prescribing of secondary cardiovascular medications. Medication costs per person for anti-diabetic, anti-obesity and cardiovascular medication were 46% higher in ROI than NI, due to differences in levels of generic prescribing.

Conclusions: These different health care systems appear to be producing similar levels of care for patients with type 2 diabetes, although at different levels of cost. The findings question the need for financial incentives in NI and highlight the large cost savings potentially accruing from a greater shift to generic prescribing in ROI. Cross-country comparison, though not without difficulties, may prove a useful adjunct to within-country analysis of policy impact.

Plasma levels of intermedin (adrenomedullin-2) in healthy human volunteers and patients with heart failure

Intermedin/adrenomedullin-2 (IMD) is a member of the adrenomedullin/CGRP peptide family. Less is known about the distribution of IMD than for other family members within the mammalian cardiovascular system, particularly in humans. The aim was to evaluate plasma IMD levels in healthy subjects and patients with chronic heart failure. IMD and its precursor fragments, preproIMD25–56 and preproIMD57–92, were measured by radioimmunoassay in 75 healthy subjects and levels of IMD were also compared to those of adrenomedullin (AM) and mid-region proadrenomedullin45–92 (MRproAM45–92) in 19 patients with systolic heart failure (LVEF < 45%). In healthy subjects, plasma levels (mean + SE) of IMD (6.3 + 0.6 pg ml−1) were lower than, but correlated with those of AM (25.8 + 1.8 pg ml−1; r = 0.49, p < 0.001). Plasma preproIMD25–56 (39.6 + 3.1 pg ml−1), preproIMD57–92 (25.9 + 3.8 pg ml−1) and MRproAM45–92 (200.2 + 6.7 pg ml−1) were greater than their respective bioactive peptides. IMD levels correlated positively with BMI but not age, and were elevated in heart failure (9.8 + 1.3 pg ml−1, p < 0.05), similarly to MRproAM45–92 (329.5 + 41.9 pg ml−1, p < 0.001) and AM (56.8 + 10.9 pg ml−1, p < 0.01). IMD levels were greater in heart failure patients with concomitant renal impairment (11.3 + 1.8 pg ml−1) than those without (6.5 + 1.0 pg ml−1; p < 0.05). IMD and AM were greater in patients receiving submaximal compared with maximal heart failure drug therapy and were decreased after 6 months of cardiac resynchronization therapy. In conclusion, IMD is present in the plasma of healthy subjects less abundantly than AM, but is similarly correlated weakly with BMI. IMD levels are elevated in heart failure, especially with concomitant renal impairment, and tend to be reduced by high intensity drug or pacing therapy.

Endothelium-derived intermedin/adrenomedullin-2 protects human ventricular cardiomyocytes from ischaemia-reoxygenation injury predominantly via the AM1 receptor

Application of intermedin/adrenomedullin-2 (IMD/AM-2) protects cultured human cardiac vascular cells and fibroblasts from oxidative stress and simulated ischaemia-reoxygenation injury (I-R), predominantly via adrenomedullin AM1 receptor involvement; similar protection had not been investigated previously in human cardiomyocytes (HCM). Expression of IMD, AM and their receptor components was studied in HCM. Receptor subtype involvement in protection by exogenous IMD against injury by simulated I-R was investigated using receptor component-specific siRNAs. Direct protection by endogenous IMD against HCM injury, both as an autocrine factor produced in HCM themselves and as a paracrine factor released from HCMEC co-cultured with HCM, was investigated using peptide-specific siRNA for IMD. IMD, AM and their receptor components (CLR, RAMPs1-3) were expressed in HCM. IMD 1 nmol L−1, applied either throughout ischaemia (3 h) and re-oxygenation (1 h) or during re-oxygenation (1 h) alone, attenuated HCM injury (P < 0.05); cell viabilities were 59% and 61% respectively vs. 39% in absence of IMD. Cytoskeletal disruption, protein carbonyl formation and caspase activity followed similar patterns. Pre-treatment (4 days) of HCM with CLR and RAMP2 siRNAs attenuated (P < 0.05) protection by exogenous IMD. Pre-treatment of HCMEC with IMD (and AM) siRNA augmented (P < 0.05) I-R injury: cell viabilities were 22% (and 32%) vs. 39% untreated HCMEC. Pre-treatment of HCM with IMD (and AM) siRNA did not augment HCM injury: cell viabilities were 37% (and 39%) vs. 39% untreated HCM. Co-culture with HCMEC conferred protection from injury on HCM; such protection was attenuated when HCMEC were pre-treated with IMD (but not AM) siRNA before co-culture. Although IMD is present in HCM, IMD derived from HCMEC and acting in a paracrine manner, predominantly via AM1 receptors, makes a marked contribution to cardiomyocyte protection by the endogenous peptide against acute I-R injury.

Smoking cessation and the incidence of pre-diabetes and type 2 diabetes: a cohort study.

AIMS: Smoking cessation has been suggested to increase the short-term risk of type 2 diabetes mellitus (T2DM). This study aimed at assessing the association between smoking cessation and incidence of T2DM and impaired fasting glucose (IFG). METHODS: Data from participants in the CoLaus study, Switzerland, aged 35-75 at baseline and followed for 5.5years were used. Participants were classified as smokers, recent (≤5years), long-term (>5years) quitters, and non-smokers at baseline. Outcomes were IFG (fasting serum glucose (FSG) 5.6-6.99mmol/l) and T2DM (FSG ≥7.0mmol/l and/or treatment) at follow up. RESULTS: 3,166 participants (63% women) had normal baseline FSG, of whom 26.7% were smokers, 6.5% recent quitters, and 23.5% long-term quitters. During follow-up 1,311 participants (41.4%) developed IFG (33.6% women, 54.7% men) and 47 (1.5%) developed T2DM (1.1% women, 2.1% men). Former smokers did not have statistically significant increased odds of IFG compared with smokers after adjustment for age, education, physical activity, hypercholesterolemia, hypertension and alcohol intake, with OR of 1.29 [95% confidence interval 0.94-1.76] for recent quitters and 1.03 [0.84-1.27] for long-term quitters. Former smokers did not have significant increased odds of T2DM compared with smokers with multivariable-adjusted OR of 1.53 [0.58-4.00] for recent quitters and 0.64 [0.27-1.48] for long-term quitters. Adjustment for body-mass index and waist circumference attenuated the association between recent quitting and IFG (OR 1.07 [0.78-1.48]) and T2DM (OR 1.28 [0.48-3.40]. CONCLUSION: In this middle-aged population, smoking cessation was not associated with an increased risk of IFG or T2DM.

The cloning and reconstitution of a bovine adenovirus type 2 E3 deletion mutant and the sequencing and analysis of the early 4 region

Recombinant Adenoviruses (Ads) have been shown to have potential applications in three areas: gene therapy, high level protein expression and recombinant vaccines.' At least three different locations within the Ad genome can be deleted and subsequently used for the insertion of foreign sequences. These include the Early 3 (E3), Early 1 (E1) and Early 4 (E4) regions. Viral vectors of this type have been well studied in Human Ads 2 and 5, however one has not yet been constructed for Bovine Adenovirus Type 2 (BAV2). The E3 region is located between 76.6 and 86 m.u. on the r-strand and is transcribed in a rightward direction. The gene products of the Early 3 region (E3) have been shown to be non-essential for viral replication, in vitro, but are required for host immunosurveillance. This study represents the cloning and reconstitution of a BAV2 E3 deletion mutant. A deletion of 1800bp was made within the E3 region of BAV2 and the thymidine kinase gene was subsequently inserted in the deleted area . . The plasmid pdlE3-4tk1 (23.4Kbp) was constructed and used to to facilitate homologous recombination with the wild type BAV2 to produce a mutant. Southern Blotting and Hybridization results suggest the presence of a BAV2 E3 deletion mutant with thymidine kinase sequences present. The E4 region of Human Adenovirus types 2 and 5 is located at the extreme right end of the genome (91.3 map units - 99.1 map units) and is transcribed in a leftward direction giving rise to a complicated set of differentially spliced mRNAs. Essentially there are 7 open reading frames (ORFs) encoding for at least 7 polypeptides. The gene products encoded by the E4 region have been shown to be essential for the expression of late viral genes, host cell shutoff and normal viral growth. We have cloned and sequenced the right end segment between 90.5 map units and 100 map units of the BAV2 genome. The results show several open reading frames which encode polypeptides exhibiting homology to three polypeptides encoded by the E4 region of human adenovirus type 2. These include the 14kDa protein encoded by ORF1, the 34kDa protein encoded by ORF6 and the 13kDa protein encoded by ORF3. The nucleotide sequence, restriction enzyme map, and ORF map of the E4 region could be very useful in future molecular manipulation of this region and could possibly explain the slow growth rate of BAV2 in MDBK cells.

La educación de postgrado en la U.A.N.L. autoestudio 1975-76

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