Agonist-dependent internalization of D2 receptors: imaging quantification by confocal microscopy

In positron emission tomography and single photon emission computed tomography studies using D2 dopamine (DA) receptor radiotracers, a decrease in radiotracer binding potential (BP) is usually interpreted in terms of increased competition with synaptic DA. However, some data suggest that this signal may also reflect agonist (DA)-induced increases in D2 receptor (D2R) internalization, a process which would presumably also decrease the population of receptors available for binding to hydrophilic radioligands. To advance interpretation of alterations in D2 radiotracer BP, direct methods of assessment of D2R internalization are required. Here, we describe a confocal microscopy-based approach for the quantification of agonist-dependent receptor internalization. The method relies upon double-labeling of the receptors with antibodies directed against intracellular as well as extracellular epitopes. Following agonist stimulation, DA D2R internalization was quantified by differentiating, in optical cell sections, the signal due to the staining of the extracellular from intracellular epitopes of D2Rs. Receptor internalization was increased in the presence of the D2 agonists DA and bromocriptine, but not the D1 agonist SKF38393. Pretreatment with either the D2 antagonist sulpiride, or inhibitors of internalization (phenylarsine oxide and high molarity sucrose), blocked D2-agonist induced receptor internalization, thus validating this method in vitro. This approach therefore provides a direct and streamlined methodology for investigating the pharmacological and mechanistic aspects of D2R internalization, and should inform the interpretation of results from in vivo receptor imaging studies.

Agonist-dependent internalization of D2 receptors: Imaging quantification by confocal microscopy

In positron emission tomography and single photon emission computed tomography studies using D2 dopamine (DA) receptor radiotracers, a decrease in radiotracer binding potential (BP) is usually interpreted in terms of increased competition with synaptic DA. However, some data suggest that this signal may also reflect agonist (DA)-induced increases in D2 receptor (D2R) internalization, a process which would presumably also decrease the population of receptors available for binding to hydrophilic radioligands. To advance interpretation of alterations in D2 radiotracer BP, direct methods of assessment of D2R internalization are required. Here, we describe a confocal microscopy-based approach for the quantification of agonist-dependent receptor internalization. The method relies upon double-labeling of the receptors with antibodies directed against intracellular as well as extracellular epitopes. Following agonist stimulation, DA D2R internalization was quantified by differentiating, in optical cell sections, the signal due to the staining of the extracellular from intracellular epitopes of D2Rs. Receptor internalization was increased in the presence of the D2 agonists DA and bromocriptine, but not the D1 agonist SKF38393. Pretreatment with either the D2 antagonist sulpiride, or inhibitors of internalization (phenylarsine oxide and high molarity sucrose), blocked D2-agonist induced receptor internalization, thus validating this method in vitro. This approach therefore provides a direct and streamlined methodology for investigating the pharmacological and mechanistic aspects of D2R internalization, and should inform the interpretation of results from in vivo receptor imaging studies.

Improved fluorescent proteins for single-molecule research in molecular tracking and co-localization

Three promising variants of autofluorescent proteins have been analyzed photophysically for their proposed use in single-molecule microscopy studies in living cells to compare their superiority to other fluorescent proteins previously reported regarding the number of photons emitted. The first variant under investigation the F46L mutant of eYFP has a 10% greater photon emission rate and > 50% slower photobleaching rate on average than the standard eYFP fluorophore. The monomeric red fluorescent protein (mRFP) has a fivefold lower photon emission rate, likely due to the monomeric content, and also a tenfold faster photobleaching rate than the DsRed fluorescent protein. In contrast, the previously reported eqfp611 has a 50% lower emission rate yet photobleaches more than a factor 2 slowly. We conclude that the F46L YFP and the eqfp611 are superior new options for single molecule imaging and tracking studies in living cells. Studies were also performed on the effects of forced quenching of multiple fluorescent proteins in sub-micrometer regions that would show the effects of dimerization at low concentration levels of fluorescent proteins and also indicate corrections to stoichiometry patterns with fluorescent proteins previously in print. We also introduce properties at the single molecule level of new FRET pairs with combinations of fluorescent proteins and artificial fluorophores.

Three dimensional analysis of cardiovascular development in mouse embryos using X-ray microcomputed tomography

Micro-computed tomography (μCT) has been successfully used to study the cardiovascular system of mouse embryos in situ. With the use of barium as a suitable contrast agent, blood vessels have been imaged and analysed quantitatively such as blood volume and vessel sizes on embryos of ages 14.5 to 16.5 days old. The advantage of using this imaging modality is that it has provided three dimensional information whilst leaving samples intact for further study.

Using historical lesion volume data in the design of a new phase II clinical trial in acute stroke

Background and Purpose-Clinical research into the treatment of acute stroke is complicated, is costly, and has often been unsuccessful. Developments in imaging technology based on computed tomography and magnetic resonance imaging scans offer opportunities for screening experimental therapies during phase II testing so as to deliver only the most promising interventions to phase III. We discuss the design and the appropriate sample size for phase II studies in stroke based on lesion volume. Methods-Determination of the relation between analyses of lesion volumes and of neurologic outcomes is illustrated using data from placebo trial patients from the Virtual International Stroke Trials Archive. The size of an effect on lesion volume that would lead to a clinically relevant treatment effect in terms of a measure, such as modified Rankin score (mRS), is found. The sample size to detect that magnitude of effect on lesion volume is then calculated. Simulation is used to evaluate different criteria for proceeding from phase II to phase III. Results-The odds ratios for mRS correspond roughly to the square root of odds ratios for lesion volume, implying that for equivalent power specifications, sample sizes based on lesion volumes should be about one fourth of those based on mRS. Relaxation of power requirements, appropriate for phase II, lead to further sample size reductions. For example, a phase III trial comparing a novel treatment with placebo with a total sample size of 1518 patients might be motivated from a phase II trial of 126 patients comparing the same 2 treatment arms. Discussion-Definitive phase III trials in stroke should aim to demonstrate significant effects of treatment on clinical outcomes. However, more direct outcomes such as lesion volume can be useful in phase II for determining whether such phase III trials should be undertaken in the first place. (Stroke. 2009;40:1347-1352.)

Automatic X-ray screening of aircraft hold luggage

R. Benjamin (1995) addressed the application of the “object 3D” X ray reconstruction technique for electronically “unpacking” suspect items, when screening aircraft luggage. However, there is no satisfactory solution to the mass screening of hold luggage. Computed Tomography, CT, entails excessive radiation dosages, and its rate of throughput is quite inadequate. A novel variant of “object 3D” is therefore put forward, adapting some of the technology of existing cabin luggage screening systems-but on a substantially larger scale-which does achieve the required throughput at an acceptable radiation dosage and cost.

Effect of polymorphisms in the PPARGC1A gene on body fat in Asian Indians

OBJECTIVE: To evaluate whether polymorphisms in the peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PPARGC1A) gene were related to body fat in Asian Indians. METHODS: Three polymorphisms of PPARGC1A gene, the Thr394Thr, Gly482Ser and +A2962G, were genotyped on 82 type 2 diabetic and 82 normal glucose tolerant (NGT) subjects randomly chosen from the Chennai Urban Rural Epidemiology Study using PCR-RFLP, and the nature of the variants were confirmed using direct sequencing. Linkage disequilibrium (LD) was estimated from the estimates of haplotypic frequencies using an expectation-maximization algorithm. Visceral, subcutaneous and total abdominal fat were measured using computed tomography, whereas dual X-ray absorptiometry was used to measure central abdominal and total body fat. RESULTS: None of the three polymorphisms studied were in LD. The genotype (0.59 vs 0.32, P=0.001) and allele (0.30 vs 0.17, P=0.007) frequencies of Thr394Thr polymorphism were significantly higher in type 2 diabetic subjects compared to those in NGT subjects. The odds ratio for diabetes (adjusted for age, sex and body mass index) for the susceptible genotype, XA (GA+AA) of Thr394Thr polymorphism, was 2.53 (95% confidence intervals: 1.30-5.04, P=0.009). Visceral and subcutaneous fat were significantly higher in NGT subjects with XA genotype of the Thr394Thr polymorphism compared to those with GG genotype (visceral fat: XA 148.2+/-46.9 vs GG 106.5+/-51.9 cm(2), P=0.001; subcutaneous fat: XA 271.8+/-167.1 vs GG 181.5+/-78.5 cm(2), P=0.001). Abdominal (XA 4521.9+/-1749.6 vs GG 3445.2+/-1443.4 g, P=0.004), central abdominal (XA 1689.0+/-524.0 vs GG 1228.5+/-438.7 g, P<0.0001) and non-abdominal fat (XA 18763.8+/-8789.4 vs GG 13160.4+/-4255.3 g, P<0.0001) were also significantly higher in the NGT subjects with XA genotype compared to those with GG genotype. The Gly482Ser and +A2962G polymorphisms were not associated with any of the body fat measures. CONCLUSION: Among Asian Indians, the Thr394Thr (G --> A) polymorphism is associated with increased total, visceral and subcutaneous body fat.

Visceral and central abdominal fat and anthropometry in relation to diabetes in Asian Indians

OBJECTIVE: The objective of the study was to examine body fat distribution using computed tomography (CT), dual-energy X-ray absorptiometry (DEXA), and anthropometry in relation to type 2 diabetes in urban Asian Indians. RESEARCH DESIGN AND METHODS: This is a case-control study of 82 type 2 diabetic and 82 age- and sex-matched nondiabetic subjects from the Chennai Urban Rural Epidemiology Study, an ongoing epidemiological study in southern India. Visceral, subcutaneous, and total abdominal fat were measured using CT, while DEXA was used to measure central abdominal and total body fat. Anthropometric measures included BMI, waist circumference, sagittal abdominal diameter (SAD), and waist-to-hip ratio. RESULTS: Visceral and central abdominal fat showed a strong correlation with each other (P <0.0001), and kappa analysis revealed a fairly good agreement between tertiles of visceral and central abdominal fat (kappa=0.44, P <0.0001). Diabetic subjects had significantly higher visceral (P=0.005) and central abdominal (P=0.011) fat compared with nondiabetic subjects. Waist circumference and SAD showed a strong correlation with visceral (P <0.01) and central abdominal (P <0.0001) fat in both diabetic and nondiabetic subjects. Logistic regression analysis revealed visceral (odds ratio [OR] 1.011, P=0.004) and central abdominal (OR 1.001, P=0.013) fat to be associated with diabetes, even after adjusting for age and sex. CONCLUSIONS: Visceral and central abdominal fat showed a strong association with type 2 diabetes. Both measures correlated well with each other and with waist circumference and SAD in diabetic and nondiabetic urban Asian Indians.