Targeting papez circuit for cognitive dysfunction- insights into deep brain stimulation for Alzheimer's disease.

Hippocampus is the most widely studied brain area coupled with impairment of memory in a variety of neurological diseases and Alzheimer's disease (AD). The limbic structures within the Papez circuit have been linked to various aspects of cognition. Unfortunately, the brain regions that include this memory circuit are often ignored in terms of understanding cognitive decline in these diseases. To properly comprehend where cognition problems originate, it is crucial to clarify any aberrant contributions from all components of a specific circuit -on both a local and a global level. The pharmacological treatments currently available are not long lasting. Deep Brain Stimulation (DBS) emerged as a new powerful therapeutic approach for alleviation of the cognitive dysfunctions. Metabolic, functional, electrophysiological, and imaging studies helped to find out the crucial nodes that can be accessible for DBS. Targeting these nodes within the memory circuit produced significant improvement in learning and memory by disrupting abnormal circuit activity and restoring the physiological network. Here, we provide an overview of the neuroanatomy of the circuit of Papez along with the mechanisms and various deep brain stimulation targets of the circuit structures which could be significant for improving cognitive dysfunctions in AD.

Treatment of H. pylori infection and gastric ulcer: Need for novel Pharmaceutical formulation.

Peptic ulcer disease (PUD) is one of the most prevalent gastro intestinal disorder which often leads to painful sores in the stomach lining and intestinal bleeding. Untreated Helicobacter pylori (H. pylori) infection is one of the major reasons for chronic PUD which, if left untreated, may also result in gastric cancer. Treatment of H. pylori is always a challenge to the treating doctor because of the poor bioavailability of the drug at the inner layers of gastric mucosa where the bacteria resides. This results in ineffective therapy and antibiotic resistance. Current treatment regimens available for gastric ulcer and H. pylori infection uses a combination of multiple antimicrobial agents, proton pump inhibitors (PPIs), H2-receptor antagonists, dual therapy, triple therapy, quadruple therapy and sequential therapy. This polypharmacy approach leads to patient noncompliance during long term therapy. Management of H. pylori induced gastric ulcer is a burning issue that necessitates alternative treatment options. Novel formulation strategies such as extended-release gastro retentive drug delivery systems (GRDDS) and nanoformulations have the potential to overcome the current bioavailability challenges. This review discusses the current status of H. pylori treatment, their limitations and the formulation strategies to overcome these shortcomings. Authors propose here an innovative strategy to improve the H. pylori eradication efficiency.

A retrospective review of a 2-year strong antimicrobial stewardship program in a tertiary care institute in Mumbai.

BACKGROUND INFORMATION: Many institutes have implemented a strict antimicrobial stewardship (AMS) program in the postantibiotic era. AIM: To investigate how the resistance pattern changes after implementation of a stringent AMS programme. METHODOLOGY: It employs a defined daily dose methodology (DDD). The formulae listed below are used to compute this for two periods: October 2015 to October 2017 (Period 1) and October 2017 to October 2019 (Period 2) (Period 2). DDD = Antibiotics used in total (g) per year The length of stay was determined using the data from the hospital's information system (HIS). The patterns of resistance to the limited antibiotics are vancomycin, linezolid, tigecycline, and colistin. In both Periods 1 and 2, skin and soft-tissue infections, urinary tract infections, bloodstream infections, and respiratory tract infections were studied in both periods. RESULTS: In the year from October 2015 to October 2017, 4569 patients received limited antibiotics out of a total of 14,544 admissions. The average length of stay was 7.48 days in Period 1, however, it was reduced to 3.96 days in Period 2 out of 15,199 patients. In vitro isolate sensitivities to vancomycin, linezolid, tigecycline, and colistin were increased. CONCLUSION: Some of the most common antibiotics were used less frequently. This appears to be linked to a shorter stay in the hospital and increased antibiotic susceptibility.

A Sensitive Spectrofluorimetric Method for Curcumin Analysis.

Curcumin (CUR), a natural polyphenolic compound extracted from the rhizomes of Curcuma longa, is used as a pharmaceutical agent, spice in food, and as a dye. Currently, CUR is being investigated for cancer treatment in Phase-II clinical trials. CUR also possesses excellent activities like anti-inflammatory, anti-microbial, and anti-oxidant, therefore quality control is crucial. The present research work was to develop a new, simple, validated and time-saving rapid 96-well plate spectrofluorimetric method for the determination of CUR. The developed method was compared with routinely used high performance liquid chromatography (HPLC) technique. The developed method were found to be linear in the concentration range of 15 to 3900 ng/mL with R2 ≥ 0.9983 for spectrofluorimetric and 50-7500 ng/mL with R2 ≥ 0.9999 for HPLC method. Accuracy, intraday and interday precision was adequate, with RSD lower than the suggested limits. The limits for the detection and the quantification of CUR were 7 and 15 ng/mL for spectrofluorimetric, and 25 and 50 ng/mL for HPLC respectively. The Bland-Altman analysis demonstrated the similarities between the two methods. The 96-well plate method was successfully applied to determine CUR in solid lipid nanoparticles (SLNs) and chitosan nanoparticles (Chi-NPs). The developed spectrofluorimetric method can hence serve as a possible replacement for the HPLC method for the quantification of CUR in healthcare and food products.

Developing an Educational Intervention to Address Financial Hardship in Cancer Patients.

OBJECTIVE: To develop an educational intervention to empower patients to manage their financial health better. PARTICIPANTS AND METHODS: This study was conducted from September 1, 2017, to January 31, 2019. Focus groups were held with social workers, case managers, and patient financial service staff and interviews were conducted with patients and caregivers to inform the content, delivery format, and timing of an intervention for mitigating financial hardship from treatment (phase 1). Based on qualitative data, theories of adult learning, and a review of the literature, we created an educational presentation to be delivered in a classroom setting. Two patient focus groups were then held for feedback on the presentation (phase 2). RESULTS: In phase 1, both patients and allied health care staff providers believed that an educational intervention about financial aspects of care early during treatment would help them cope and plan better. Participants' suggestions for the intervention's content included billing information, insurance, authorization processes, employment policies related to health care and disability benefits, and alternative financial resources. Based on these suggestions, a preliminary educational presentation was developed with 3 main themes: insurance issues, employment issues, and financial health. Phase 2 focus group participants suggested refinement of the presentation, including targeting specific groups, adding graphics, and more information about resources. CONCLUSION: Our study provides the basis for a patient-centered education module for emotional, instrumental, and informational support for financial distress for use in a clinical setting.

Red Emitting Hydroxybenzazole (HBX) Based Azo Dyes: Linear and Non Linear Optical Properties, Optical Limiting, Z Scan Analysis with DFT Assessments.

Herein, we report the hydroxybenzazole (HBX) containing azo dyes for "linear and non-linear optical" (NLO) applications. These bi-heterocyclic dyes have HBX scaffold (decorated with ESIPT core) and connected to another thiazole moietiy through azo bond. In DMF and DMSO, dyes are "emissive in yellow-red region" and "large Stokes shift" in the range of 62-121 nm were observed. "Nonlinear absorptive coefficient" (ß), "nonlinear refractive index" (ƞ2), "third order non-linear optical susceptibility" (χ3) in DMSO, ethanol and methanol were calculated using simple and effective "Z-scan technique" having "Nd: YAG laser" at 532 nm wavelength. 4.46 × 10-13 (e.s.u.) was the highest (χ3) was observed in DMSO among all the dyes. Optical Limiting (OL) values are in the range of 7.61-19.06 J cm-2 in solvents. Thermo Gravimetric Analysis (TGA) supports that, these compounds are useful for numerous high-temperature practices in the construction of electronic as well as optical devices. Band gap was calculated by CV as well as by DFT in acetonitrile. The same trend was observed when these HOMO-LUMO gaps were correlated in between CV and DFT. To gain more insights into structural parameters, molecular geometries were optimized at "B3LYP-6-311 + G (d,p)" level of theory. Further, "Molecular Electrostatic Potential" (MEP), "Frontier Molecular Orbitals" (FMO) were presented using "Density Functional Theory (DFT)". Global hybrid functional (B3LYP, BHandHLYP) and range separated hybrid functionals (RSH) i.e. CAM-B3LYP, ωB97, ωB97X, and ωB97XD were used to calculate linear and NLO properties. Graphical Abstract.

Biomarker profiling of vitamin responsive seizures: a potential tool to detect pediatric seizures of unknown aetiology.

Aim: Certain rare inborn errors of metabolism clinically present with intractable seizures that readily respond to vitamin therapy. If identified early, brain damage due to seizures can be prevented. Methodology: A LC-MS method was developed and validated for the simultaneous quantification of the biomarkers in selected vitamin responsive pediatric seizures from dried blood spots. Results: Application of the validated method to a seizure cohort of 46 patients indicated strong agreement of the method for clinical validity. Reference intervals for these biomarkers in dried blood spots were also determined for the population, after screening 956 neonates. Conclusion: The developed method was seen to be sensitive, linear, accurate and precise for testing vitamin responsive pediatric seizures.

Identification of variant HIV envelope proteins with enhanced affinities for precursors to anti-gp41 broadly neutralizing antibodies.

HIV envelope protein (Env) is the sole target of broadly neutralizing antibodies (BNAbs) that are capable of neutralizing diverse strains of HIV. While BNAbs develop spontaneously in a subset of HIV-infected patients, efforts to design an envelope protein-based immunogen to elicit broadly neutralizing antibody responses have so far been unsuccessful. It is hypothesized that a primary barrier to eliciting BNAbs is the fact that HIV envelope proteins bind poorly to the germline-encoded unmutated common ancestor (UCA) precursors to BNAbs. To identify variant forms of Env with increased affinities for the UCA forms of BNAbs 4E10 and 10E8, which target the Membrane Proximal External Region (MPER) of Env, libraries of randomly mutated Env variants were expressed in a yeast surface display system and screened using fluorescence activated cell sorting for cells displaying variants with enhanced abilities to bind the UCA antibodies. Based on analyses of individual clones obtained from the screen and on next-generation sequencing of sorted libraries, distinct but partially overlapping sets of amino acid substitutions conferring enhanced UCA antibody binding were identified. These were particularly enriched in substitutions of arginine for highly conserved tryptophan residues. The UCA-binding variants also generally exhibited enhanced binding to the mature forms of anti-MPER antibodies. Mapping of the identified substitutions into available structures of Env suggest that they may act by destabilizing both the initial pre-fusion conformation and the six-helix bundle involved in fusion of the viral and cell membranes, as well as providing new or expanded epitopes with increased accessibility for the UCA antibodies.

Development and validation of a dried blood spot test for thiamine deficiency among infants by HPLC-fluorimetry.

Thiamine deficiency, if detected early in infancy, can be treated with thiamine supplementation and can prevent seizures, other disabilities and death. The dried blood spot (DBS) sampling technique is an attractive sample collection technique for infants. The present study reports the development and validation of a highly sensitive and precise method for quantification of thiamine diphosphate from DBS. The method utilizes full-spot analysis of a volumetrically deposited 40 µl DBS. The analyte was extracted from the DBS using 50% methanol and then derivatized using potassium ferricyanide to thiochrome. Separation was achieved with the help of an Inertsil ODS C18 column (5.0 µm, 250 × 4.6 mm) using 150 mm phosphate buffer pH 7-acetonitrile (90:10, % v/v) as the mobile phase. The use of a fluorimetric detector gave a good response to the thiochrome derivative offering good sensitivity for the method. The excitation and emission wavelengths were 367 and 435 nm, respectively. The limit of detection and lower limit of quantification were 5 and 10 ng/ml, respectively. Linearity was demonstrated from 10 to 1000 ng/ml, and precision (CV) was <12.08%, at all tested quality control levels. The method accuracy was 89.34-118.89% with recoveries >80%. Bland-Altman analysis of DBS sampling vs. whole blood demonstrated a mean bias of only 1.16 ng/ml, with a majority of the 60 investigated patient samples lying within 7.2% of the corresponding concentration measured in blood, thereby meeting the clinical desirable biological specification criterion and showing that the two methods are comparable.

Novel HILIC-ESI-MS method for urinary profiling of MSUD and methylmalonic aciduria biomarkers.

Methyl malonic acid and branched-chain keto acids are important biomarkers for the diagnosis of cobalamin deficiencies and maple syrup urine disease. We report the development and validation of a HILIC-ESI-MS2 method for the quantification of these organic acids from neonatal urine. The samples were 100 times diluted and analyzed on a ZIC-HILIC column with 25-mM formic acid in water: 25-mM formic acid in acetonitrile (45:55) at a flow rate of 0.8 mL/min with a runtime of only 6 minutes. The method demonstrated a lower limit of detection of 10 ng/mL, Limit of Quantification (LOQ) of 50 ng/mL, linearity of r2 ≥ 0.990 and recoveries of 87-105% for all analytes. The intraday and interday precision CV's were <10% and 12%, respectively. Extensive stability studies demonstrated the analytes to be stable in stock and in matrix with a percent change within ±15%. The Bland-Altman analysis of the developed method with the gold standard GCMS method demonstrated a bias of 0.44, 0.11, 0.009 and -0.19 for methyl malonic acid, 3-methyl-2-oxovaleric acid, 2-hydroxy-3methylbutyric acid and 4-methyl-2-oxovaleric acid, respectively, proving the methods are comparable. The newly developed method involves no derivatization and has a simple sample preparation and a low runtime, enabling it to be easily automated with a high sample throughput in a cost-effective manner.

Cost-Effective HPLC-UV Method for Quantification of Vitamin D2 and D3 in Dried Blood Spot: A Potential Adjunct to Newborn Screening for Prophylaxis of Intractable Paediatric Seizures.

25-Hydroxyvitamin D (25-(OH)D) deficiency is recently been described as one of the multiple factors responsible for pediatric seizures. 25-Hydroxyvitamin D3 and 25-Hydroxyvitamin D2 are the well-known markers to determine Vitamin D status. In this work we report the development of a sensitive and cost effective HPLC technique for the quantification of the vitamin D metabolites from dried blood spot samples (DBS). The metabolites were extracted using acetonitrile-methanol-0.1% formic acid (60 : 20 : 20 (v/v)) and analyzed on an Acclaim C18 column (150 × 4.6 mm i.d., 3 µm) at a flow rate of 1 mL/min. The method was linear in the range of 10-80 ng/mL. Limit of detection and limit of quantification (LOQ) of the method were 5 and 10 ng/mL respectively. Extensive stability studies demonstrated the analytes to be stable in stock and matrix with a percent change within the acceptable range of ±15%. Comparison of the newly developed HPLC-DBS method with the reported LC-MS-DBS and electrochemiluminescence immunoassay (ECLIA) methods followed by Bland-Altman analysis demonstrated a bias of 0.08 and -0.14, respectively proving the methods are comparable. Application of the developed method to a pediatric seizure cohort depicted 46.6% of cases as deficient and 26.6% as insufficient for 25-(OH)D. Among deficient cases 8 samples were below 10 ng/mL and exact amount was not calculated since these were below the LOQ levels. The mean ± standard deviation (S.D.) in the remaining 6 deficient cases was 13.22 ± 2.80 ng/mL. The levels in healthy infants were 33.9 ± 6.11 ng/mL. The method can be used routinely for assessing 25-(OH)D deficiency in newborn.

NLOphoric Triphenylamine Derived Donor-π-Acceptor-π-Donor Based Colorants: Synthesis, Spectroscopic, Density Functional Theory and Z-scan Studies.

Three Donor-π-Acceptor-π-Donor type styryl dyes (5a-c) with different secondary donors are synthesized and characterized to study their nonlinear and linear optical properties. The structure-property relationships of the dyes are described in the light of systematic photophysical and theoretical investigations. The photophysical characteristics of 5a-c are influenced by the polarity of the medium, with an appreciable bathochromic shift in emission (5b = 81 nm) and large Stoke shifts (5b = 104-173 nm) in polar solvents. 5a-c showed intramolecular charge transfer characteristics recognized with the help of emission solvatochromism, solvent polarity graphs, natural bond orbital analysis and HOMO-LUMO energy difference. The optimized geometry and frontier molecular orbitals reveal that the electron donation takes place from secondary donors and not from a fixed donor (triphenylamine) which is more twisted. The nonlinear optical properties obtained using solvent induced spectral shift and computational methods are found within the limiting values. Z-scan results reveal saturable kind of behavior for 5a, 5b and 5c, whereas 5a and 5b show reverse saturable kind of behavior in acetone and ethanol and hence give optical limiting values. The two-photon absorption cross section described by two-level approximation is highest for 5b (251-300 GM).

Display of the HIV envelope protein at the yeast cell surface for immunogen development.

As a step toward the development of variant forms of Env with enhanced immunogenic properties, we have expressed the glycoprotein in the yeast surface display system in a form that can be subjected to random mutagenesis followed by screening for forms with enhanced binding to germline antibodies. To optimize the expression and immunogenicity of the yeast-displayed Env protein, we tested different approaches for cell wall anchoring, expression of gp120 and gp140 Env from different viral strains, the effects of introducing mutations designed to stabilize Env, and the effects of procedures for altering N-linked glycosylation of Env. We find that diverse forms of HIV envelope glycoprotein can be efficiently expressed at the yeast cell surface and that gp140 forms of Env are effectively cleaved by Kex2p, the yeast furin protease homolog. Multiple yeast-displayed gp120 and gp140 proteins are capable of binding to antibodies directed against the V3-variable loop, CD4 binding site, and gp41 membrane-proximal regions, including some antibodies whose binding is known to depend on Env conformation and N-linked glycan. Based on antibody recognition and sensitivity to glycosidases, yeast glycosylation patterns partially mimic high mannose-type N-glycosylation in mammalian cells. However, yeast-displayed Env is not recognized by some anti-Env antibodies sensitive to quaternary structure, suggesting either that the displayed protein exists in a monomeric state or that for these antibodies, yeast glycosylation in certain regions hinders recognition or access. Consistent with studies in other systems, reconstructed predicted unmutated precursors to anti-Env antibodies exhibit little affinity for the yeast-displayed envelope protein.

Biomarker Profiling for Pyridoxine Dependent Epilepsy in Dried Blood Spots by HILIC-ESI-MS.

Pyridoxine dependent epilepsy is a condition where the affected infant or child has prolonged seizures (status epilepticus), which are nonresponsive to anticonvulsant therapy but can be treated with pharmacological doses of pyridoxine. If identified earlier and treated prophylactically with pyridoxine, severe brain damage due to seizures can be prevented. Alpha-amino adipic semialdehyde (AASA), piperidine-6-carboxylic acid (P6C), and pipecolic acid (PA) are known biomarkers of pyridoxine dependent epilepsy. We report the development and validation of a hydrophilic interaction liquid chromatography (HILIC) hyphenated with mass spectroscopy for the quantification of the above analytes from dried blood spot samples. The samples were extracted using methanol and analysed on a iHILIC fusion plus column with formic acid buffer (pH 2.5): acetonitrile (20:80) at a flow rate of 0.5 mL/min within 3 minutes. The method demonstrated a LOD of 10 ng/mL, LOQ of 50 ng/mL, linearity of r2 ≥ 0.990, and recovery of 92-101.98% for all analytes. The intra- and interday precision CVs were < 8% and 6%, respectively. Extensive stability studies demonstrated that the analytes were stable in stock solution and in matrix when stored at -80°C. We performed method comparison studies of the developed method with the literature reported method using normal samples and matrix matched spiked samples at pathological concentrations to mimic clinical validity. The Bland-Altman analysis for comparison of the analytical suitability of the method for the biomarkers in healthy and spiked samples with the literature reported method revealed a bias which suggested that the method was comparable. The newly developed method involves no derivatisation and has a simple sample preparation and a low run time enabling it to be easily automated with a high sample throughput in a cost-effective manner.

A migratory shark bone.

Fish bone ingestion is a common presentation in ENT. If not managed correctly, it can cause serious complications for the patient and dilemmas for the clinician. A 49-year-old Sri Lankan woman presented to the emergency department following shark bone ingestion with a 'pricking' sensation in her throat. After initial investigation, the bone migrated through to the sternocleidomastoid muscle. After surgical removal of the shark bone she went on to develop a large neck collection, which required surgical drainage. The careful attention to the patient's history and use of imaging facilitated treatment in this case of fish bone ingestion and management of the sequelae.

A Novel Screening Approach for Optimal and Functional Fusion of T4 Lysozyme in GPCRs.

Determination of high-resolution, three-dimensional structures of transmembrane proteins (TMPs) has, in many cases, only been accomplished through the use of stabilized variant forms of the proteins being studied. For the important G protein-coupled receptor superfamily, this has most often been achieved by inserting a stable soluble protein, such as T4 lysozyme (T4L), in an internal loop of a receptor. However, creation of such fusion proteins generally results in loss of the ability of receptors to activate their cognate cytoplasmic G proteins. Furthermore, the criteria for designing fusions that minimally perturb receptor structure are not well established. We describe here a method for creating a library of receptor variants containing T4L inserted into an internal loop at varying positions and as replacements for varying amounts of the original receptor sequence. We also describe methods for screening for variants displaying maximal expression levels, ligand binding capacity, and signaling function. When applied to the yeast α-factor receptor, Ste2p, this approach allowed recovery of well-expressed receptor variants containing internally fused T4L that retained nearly normal signaling function. The approach we describe can be readily adapted to creation of stabilized fusions of other TMPs expressed in yeast or other expression systems.

Chronic hyperglycemia downregulates GLP-1 receptor signaling in pancreatic ß-cells via protein kinase A.

OBJECTIVE: Glucagon-like peptide 1 (GLP-1) enhances insulin secretion and protects ß-cell mass. Diabetes therapies targeting the GLP-1 receptor (GLP-1R), expressed in numerous tissues, have diminished dose-response in patients with type 2 diabetes compared with healthy human controls. The aim of this study was to determine the mechanistic causes underlying the reduced efficacy of GLP-1R ligands. METHODS: Using primary mouse islets and the ß-cell line MIN6, outcomes downstream of the GLP-1R were analyzed: Insulin secretion; phosphorylation of the cAMP-response element binding protein (CREB); cAMP responses. Signaling systems were studied by immunoblotting and qRT-PCR, and PKA activity was assayed. Cell surface localization of the GLP-1R was studied by confocal microscopy using a fluorescein-tagged exendin-4 and GFP-tagged GLP-1R. RESULTS: Rodent ß-cells chronically exposed to high glucose had diminished responses to GLP-1R agonists including: diminished insulin secretory response; reduced phosphorylation of (CREB); impaired cAMP response, attributable to chronically increased cAMP levels. GLP-1R signaling systems were affected by hyperglycemia with increased expression of mRNAs encoding the inducible cAMP early repressor (ICER) and adenylyl cyclase 8, reduced PKA activity due to increased expression of the PKA-RIα subunit, reduced GLP-1R mRNA expression and loss of GLP-1R from the cell surface. To specifically examine the loss of GLP-1R from the plasma membrane a GLP-1R-GFP fusion protein was employed to visualize subcellular localization. Under low glucose conditions or when PKA activity was inhibited, GLP-1R-GFP was found at the plasma membrane. Conversely high glucose, expression of a constitutively active PKA subunit, or exposure to exendin-4 or forskolin led to GLP-1R-GFP internalization. Mutation of serine residue 301 of the GLP-1R abolished the glucose-dependent loss of the receptor from the plasma membrane. This was associated with a loss of an interaction between the receptor and the small ubiquitin-related modifier (SUMO), an interaction that was found to be necessary for internalization of the receptor. CONCLUSIONS: These data show that glucose acting, at least in part, via PKA leads to the loss of the GLP-1R from the cell surface and an impairment of GLP-1R signaling, which may underlie the reduced clinical efficacy of GLP-1R based therapies in individuals with poorly controlled hyperglycemia.

Guided reconstitution of membrane protein fragments.

Structural analysis by NMR of G protein-coupled receptors (GPCRs) has proven to be extremely challenging. To reduce the number of peaks in the NMR spectra by segmentally labeling a GPCR, we have developed a Guided Reconstitution method that includes the use of charged residues and Cys activation to drive heterodimeric disulfide bond formation. Three different cysteine-activating reagents: 5-5'-dithiobis(2-nitrobenzoic acid) [DTNB], 2,2'-dithiobis(5-nitropyridine) [DTNP], and 4,4'-dipyridyl disulfide [4-PDS] were analyzed to determine their efficiency in heterodimer formation at different pHs. Short peptides representing the N-terminal (NT) and C-terminal (CT) regions of the first extracellular loop (EL1) of Ste2p, the Saccharomyces cerevisiae alpha-factor mating receptor, were activated using these reagents and the efficiencies of activation and rates of heterodimerization were analyzed. Activation of NT peptides with DTNP and 4-PDS resulted in about 60% yield, but heterodimerization was rapid and nearly quantitative. Double transmembrane domain protein fragments were biosynthesized and used in Guided Reconstitution reactions. A 102-residue fragment, 2TM-tail [Ste2p(G31-I120C)], was heterodimerized with CT-EL1-tail(DTNP) at pH 4.6 with a yield of ∼75%. A 132-residue fragment, 2TMlong-tail [Ste2p(M1-I120C)], was expressed in both unlabeled and (15)N-labeled forms and used with a peptide comprising the third transmembrane domain, to generate a 180-residue segmentally labeled 3TM protein that was found to be segmentally labeled using [(15)N,(1)H]-HSQC analysis. Our data indicate that the Guided Reconstitution method would be applicable to the segmental labeling of a membrane protein with 3 transmembrane domains and may prove useful in the preparation of an intact reconstituted GPCR for use in biophysical analysis and structure determination.

Fluorescent approaches for understanding interactions of ligands with G protein coupled receptors.

G protein coupled receptors are responsible for a wide variety of signaling responses in diverse cell types. Despite major advances in the determination of structures of this class of receptors, the underlying mechanisms by which binding of different types of ligands specifically elicits particular signaling responses remain unclear. The use of fluorescence spectroscopy can provide important information about the process of ligand binding and ligand dependent conformational changes in receptors, especially kinetic aspects of these processes that can be difficult to extract from X-ray structures. We present an overview of the extensive array of fluorescent ligands that have been used in studies of G protein coupled receptors and describe spectroscopic approaches for assaying binding and probing the environment of receptor-bound ligands with particular attention to examples involving yeast pheromone receptors. In addition, we discuss the use of fluorescence spectroscopy for detecting and characterizing conformational changes in receptors induced by the binding of ligands. Such studies have provided strong evidence for diversity of receptor conformations elicited by different ligands, consistent with the idea that GPCRs are not simple on and off switches. This diversity of states constitutes an underlying mechanistic basis for biased agonism, the observation that different stimuli can produce different responses from a single receptor. It is likely that continued technical advances will allow fluorescence spectroscopy to play an important role in continued probing of structural transitions in G protein coupled receptors. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding.

The effects of taurine and thiotaurine on oxidative stress in the aorta and heart of diabetic rats.

This study has compared the actions of the sulfur-containing compounds taurine (TAU) and thiotaurine (TTAU) with those of insulin (INS) on the oxidative stress that develops in the aorta and heart as a result of diabetes. Diabetes was induced in male Sprague-Dawley rats with streptozotocin (60 mg/kg, i.p.). Starting on day 15, and continuing for the next 41 days, the diabetic rats received each day 2 mL of physiological saline or 2.4 mmol/kg/2 mL of TAU (or TTAU) p.o. or 4 U/kg of isophane INS s.c. Normal rats served as controls. The rats were sacrificed on day 57 to collect blood, heart and thoracic aorta samples. Untreated diabetic rats exhibited a lower body weight gain (by 34%), higher than normal plasma glucose (by ∼4-fold), cholesterol (by 66%) and triglycerides (by 188%) levels, and lower INS levels (by 76%). Also there was a marked increase in catalase activity (≥90%); and clear decreases in nitrite (≥40%), glutathione redox status (≥67%), and glutathione peroxidase (≥66%) and superoxide dismutase (≥51%) activities in both the aorta and heart. With only a few isolated instances (plasma lipids), TTAU was either markedly more effective (plasma glucose, plasma INS, aorta and heart glutathione, aorta redox status, and antioxidant enzymes) or marginally more effective (heart redox status) than TAU in attenuating the alterations brought about by diabetes. These results suggest that replacing the sulfonic acid group of TAU by thiosulfonic acid can lead to a greater potency against diabetes-related biochemical changes in the plasma, heart and aorta. However, except for effects on plasma lipids, these sulfur-containing compounds were less effective than INS in counteracting diabetes-related changes.