Reconstitution of affinity-purified dopamine D2 receptor binding activities by specific lipids

The role of lipids in maintaining ligand binding properties of affinity-purified bovine striatal dopamine D₂ receptor was investigated in detail. The receptor, purified on a haloperidol-linked Sepharose CL6B affinity column, exhibited low [³H]spiroperidol binding unless reconstituted with soybean phospholipids. In order to understand the role of individual phospholipids in maintaining the receptor binding activity, the purified preparation was reconstituted separately with individual phospholipids and assayed for [³H]spiroperidol binding. Except for phosphatidylcholine and phosphatidylethanolamine, that respectively restored 30 and 20% binding as compared to that obtained with soybean lipids, reconstitution with other lipids had very little effect. When various combinations of phospholipids were used for reconstitution, a phosphatidylcholine and phosphatidylserine mixture seemed to almost fully restore the receptor binding. A mixture of phosphatidylcholine and phosphatidylethanolamine was as effective as phosphatidylcholine alone in reconstituting ligand binding; however, when phosphatidylserine was also included in the mixture, there was a pronounced increase in binding (about 2-fold compared to the soybean lipids and about 6-fold compared to the phosphatidylcholine-phosphatidylethanolamine mixture). Substitution of other phospholipids or cholesterol for phosphatidylserine in phosphatidylcholine and phosphatidylethanolamine mixture had little effect. Maximal reconstitution of [³H]spiroperidol binding was obtained with phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine mixture (2:2:1, w/w) at a concentration of 0.5 mg/ml. The reconstituted receptor exhibited high affinity binding for [³H]spiroperidol which was comparable to that obtained with membrane or solubilized preparations. Various dopaminergic antagonists and agonists showed appropriate order of potency for the reconstituted receptor. The presently described reconstitution data suggest a role of specific phospholipids in preserving the binding properties of dopamine D₂ receptor and should prove useful in studies on functional reconstitution of the receptor.     

Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis

G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-L-phenylalanine (Acp) and p-benzoyl-L-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to approximately 50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5-2 microg/10(7) cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.     

Ligand dependence in the copper-catalyzed oxidation of hydroquinones

Transition metal-mediated oxidation of hydroquinones is an important physiologic reaction, and copper(II) effectively catalyzes the reaction in phosphate-buffered saline (PBS). Studies reported herein in phosphate buffer alone demonstrate that copper(II) is an ineffective catalyst in the absence of coordinating ligands, but that 1,10-phenanthroline and histamine facilitate the copper(II)-mediated oxidation of hydroquinone and its 2,5- and 2,6-di-tert-butyl analogs to the corresponding benzoquinones. The high concentration of chloride in PBS is the key element that allows copper(II) to work in this system. Although the bis-bathocuproine disulfonate complex of Cu(II), (BC)₂Cu(II), is a strong stoichiometric oxidant, stoichiometric amounts of copper(II) in the presence of ligands other than BC oxidize hydroquinones very slowly under anaerobic conditions. Thus, the rapid copper(II)-catalyzed reaction operating aerobically does not involve a simple ping-pong reduction of copper(II) to copper(I) by hydroquinone and reoxidation of copper(I) by O₂.     

Survival of patients treated with intra-aortic balloon counterpulsation at a tertiary care center in Pakistan – patient characteristics and predictors of in-hospital mortality

Background

In most western countries 20% of adults have hypertension. Reports in the literature suggest that from 31 to 86% of treated patients are not at recommended target levels. However it is important to consider how we are determining whether targets are unmet and the degree to which they are unmet. Our underlying hypothesis is that white coat effect is partially responsible for the reported low rates of control of hypertension by primary care practitioners.

Methods

The study population consists of 1142 patients who are being assessed for enrolment in two community-based randomized controlled trials. Patients must have essential hypertension, be on antihypertensive medication, and must not have met their blood pressure targets. We are reporting on the proportion of patients who have not achieved target, and the degree to which they have not achieved their target. We also report on the mean daytime blood pressures on 24 hour ABPM and compare these to mean blood pressures found on the patients' charts.

Results

We identified 3284 patient charts of patients with hypertension. Of these, 1142 were determined to be "out of control" (did not achieve target) and 436 agreed to undergo 24 hour ABPM for final determination of eligibility. Overwhelmingly (95.8% of the time) it was the systolic blood pressure that was not under control. However, most of the patients who had not achieved target according to our criteria were within 10 mmHg of the recommended targets. Isolated systolic blood pressure was the best predictor of elevated mean daytime blood pressure on 24 hour ABPM.

Conclusions

At least 35% of patients had not achieved target blood pressure levels and this is primarily due to lack of control of systolic blood pressure. The best predictor of continuing hypertension on 24 hour ABPM was the mean systolic blood pressure on the patients chart. However, only 69% of patients who were uncontrolled according blood pressures recorded in the chart were uncontrolled according to 24 hour ABPM criteria. This suggests that the white coat effect makes blood pressure measurements in the doctor's offices, at least as currently done, not sufficiently accurate for determining treatment endpoint.     

Glucose-stimulated insulin response in pregnant sheep following acute suppression of plasma non-esterified fatty acid concentrations

Background  

Elevated non-esterified fatty acids (NEFA) concentrations in non-pregnant animals have been reported to decrease pancreatic responsiveness. As ovine gestation advances, maternal insulin concentrations fall and NEFA concentrations increase. Experiments were designed to examine if the pregnancy-associated rise in NEFA concentration is associated with a reduced pancreatic sensitivity to glucose in vivo. We investigated the possible relationship of NEFA concentrations in regulating maternal insulin concentrations during ovine pregnancy at three physiological states, non-pregnant, non-lactating (NPNL), 105 and 135 days gestational age (dGA, term 147+/- 3 days).     

Selective reconstitution of human D4 dopamine receptor variants with Gi alpha subtypes

G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extracellular molecules and transduce signals by coupling to heterotrimeric G proteins in the cytoplasm. The human D4 dopamine receptor is a particularly interesting GPCR because the polypeptide loop linking TM helices 5 and 6 (loop i3) may contain from 2 to 10 similar direct hexadecapeptide repeats. The precise role of loop i3 in D4 receptor function is not known. To clarify the role of loop i3 in G protein coupling, we constructed synthetic genes for the three main D4 receptor variants. D4-2, D4-4, and D4-7 receptors contain 2, 4, and 7 imperfect hexadecapeptide repeats in loop i3, respectively. We expressed and characterized the synthetic genes and found no significant effect of the D4 receptor polymorphisms on antagonist or agonist binding. We developed a cell-based assay where activated D4 receptors coupled to a Pertussis toxin-sensitive pathway to increase intracellular calcium concentration. Studies using receptor mutants showed that the regions of loop i3 near TM helices 5 and 6 were required for G protein coupling. The hexadecapeptide repeats were not required for G protein-mediated calcium flux. Cell membranes containing expressed D4 receptors and receptor mutants were reconstituted with purified recombinant G protein alpha subunits. The results show that each D4 receptor variant is capable of coupling to several G(i)alpha subtypes. Furthermore, there is no evidence of any quantitative difference in G protein coupling related to the number of hexadecapeptide repeats in loop i3. Thus, loop i3 is required for D4 receptors to activate G proteins. However, the polymorphic region of the loop does not appear to affect the specificity or efficiency of G(i)alpha coupling.     

Alignment of high resolution mass spectra: development of a heuristic approach for metabolomics

One of the challenges of using mass spectrometry for metabolomic analyses of samples consisting of thousands of compounds is that of peak identification and alignment. This paper addresses the issue of aligning mass spectral data from different samples in order to determine average component m/z peak values. The alignment scheme developed takes the instrument m/z measurement error into consideration in order to heuristically align two or more samples using a technique comparable to automated visual inspection and alignment. The results obtained using mass spectral profiles of replicate human urine samples suggest that this heuristic alignment approach is more efficient than other approaches using hierarchical clustering algorithms. The output consists of an average m/z and intensity value for the spectral components together with the number of matches from the different samples. One of the major advantages of using this alignment strategy is that it eliminates the boundary problem that occurs when using predetermined fixed bins to identify and combine peaks for averaging and the efficient runtime allows large datasets to be processed quickly.     

Malate-aspartate shuttle enzymes in rat brain regions, liver and heart during alloxan diabetes and insulin replacement

Regionally selective and time-dependent variations were observed in the activity of brain aspartate aminotransferase at early phases of diabetes. Malate dehydrogenase activity showed an opposite pattern of changes in soluble and particulate fractions of cerebral hemispheres and brain stem, with cerebellum showing consistent increase in the activity. The activity of both the enzymes increased significantly in liver, in contrast to heart where malate dehydrogenase activity decreased in particulate fraction. Insulin treatment to diabetic animals restored the enzymes to near control levels at early stages of diabetes, except in liver. The results indicate that malate-aspartate shuttle is probably stimulated under diabetic conditions to enable glycolysis to continue and ATP levels to be restored partially, particularly in cerebellum and liver.     

Development of an in-house enzyme-linked immunosorbent assay based on surface whole cell antigen for diagnosis of Helicobacter pylori infection in patients with gastroduodenal ulcer disease

Helicobacter pylori (H. pylori) is a causative agent of gastritis, gastroduodenal ulcers and gastric adenocarcinoma. More than 50 % world population is colonized by H. pylori, which is closely related to the chronic gastritis and gastric ulcer infection. In this study, a total of 214 gastritis patient’s serum samples were screened for anti-H. pylori IgG antibody. A 96-well plate coated with 20 μg/ml antigen and hundred-fold diluted patient’s serum was allowed to react. After extensive washing with buffer, 1:2,500 diluted conjugated secondary antibody was added. Later substrate was added to observe positivity by measuring the intensity of color. Statistical analyses were performed, and p value of <0.01 was taken as significant; 84 % male patients and 89 % female patients, respectively, tested positive for H. pylori, while agewise distribution was 35–45 years males (40 %) and 35–55 years females (52 %) were found highest number of H. pylori infected patients. In-house ELISA based on surface whole cell antigen (wELISA) showed a sensitivity of 93 %, specificity of 100 %, accuracy 94 % and κ value 0.86 with significant correlation R—0.77020; p < 0.0001. We conclude that H. pylori local isolates surface antigen was satisfactory for diagnosis as different parameters were adjusted according to the local H. pylori isolates. Fluctuations in serum antibody titer predict the variation in an individual’s response of the host against H. pylori. In-house wELISA could provide a reliable and a clinically useful method for the diagnosis of H. pylori infection in patients of Karachi, Pakistan.