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Ecdysone agonists are a class of insecticides that activate the ecdysone receptor (EcR) heterodimerized with the ultraspiracle (USP). Here, we report a new luciferase reporter assay for ecdysone agonists. The assay employs mammalian HEK293T cells transiently transfected with the EcR and USP genes of Chilo suppressalis, along with the taiman (Tai) gene of Drosophila melanogaster that encodes a steroid receptor coactivator. This assay system gave results consistent with those of radioligand binding assays and showed sensitivity superior to that of the existing in vitro methods. In addition, use of the heterologous host cells precludes perturbation from intrinsic players of the ecdysone signaling, which is a potential drawback of insect cell-based methods. This reporter system is suitable for detailed structure-activity analysis of ecdysone agonists and will serve as a valuable tool for the rational design of novel insect growth regulators. [ABSTRACT FROM AUTHOR]

Purpose We report our initial clinical experience with β -emitting 177 Lu-PSMA-I&T ( 177 Lu labeled prostate specific membrane antigen ligand for imaging and therapy) for systemic treatment of metastatic castration resistant prostate cancer. Materials and Methods Patients with metastatic castration resistant prostate cancer who experienced treatment failure with chemotherapy and novel androgen receptor targeted therapy were treated for 8 weeks with up to 4 cycles of 177 Lu-PSMA-I&T. We report safety data, the antitumor response with prostate specific antigen decreases and the radiographic tumor response as well as the clinical outcome with changes in ECOG (Eastern Cooperative Oncology Group) performance status and pain severity. Results The first 3 patients were treated with a lower activity of 3.7 GBq in cycle 1. Due to a favorable safety profile the activity was increased to 7.4 GBq in 19 subsequent patients who completed a total of 40 cycles. With the higher activity no grade 3/4 toxicities were observed. The main nonhematological and hematological grade 1/2 toxicities were dry mouth in 7 patients (37%), anemia in 6 (32%) and thrombopenia in 5 (25%). The proportion of patients who achieved a maximum prostate specific antigen decrease of 30% or greater, 50% or greater and 90% or greater was 56%, 33% and 11%, respectively. Combined assessment of bone and soft tissue metastases showed complete remission in 5% of patients, stable disease in 63% and progressive disease in 32%. ECOG performance status improved or was stable in 74% of patients. Of men with bone pain 58% achieved complete resolution or reduced pain. Conclusions Radioligand therapy with 177 Lu-PSMA-I&T appears to be safe and active in heavily pretreated patients with metastatic castration resistant prostate cancer. [ABSTRACT FROM AUTHOR]

Background: Ventricular arrhythmias as a result of unintentional blockade of the Kv11.1 (hERG [human ether-à-go-go-related gene]) channel are a major safety concern in drug development. In past years, several highly prescribed drugs have been withdrawn for their ability to cause such proarrhythmia. Here, we investigated whether the proarrhythmic risk of existing drugs could be reduced by Kv11.1 allosteric modulators. Methods and Results: Using [(3)H]dofetilide-binding assays with membranes of human Kv11.1-expressing human embryonic kidney 293 cells, 2 existing compounds (VU0405601 and ML-T531) and a newly synthesized compound (LUF7244) were found to be negative allosteric modulators of dofetilide binding to the Kv11.1 channel, with LUF7244 showing the strongest effect at 10 μmol/L. The Kv11.1 affinities of typical blockers (ie, dofetilide, astemizole, sertindole, and cisapride) were significantly decreased by LUF7244. Treatment of confluent neonatal rat ventricular myocyte (NRVM) monolayers with astemizole or sertindole caused heterogeneous prolongation of action potential duration and a high incidence of early afterdepolarizations on 1-Hz electric point stimulation, occasionally leading to unstable, self-terminating tachyarrhythmias. Pretreatment of NRVMs with LUF7244 prevented these proarrhythmic effects. NRVM monolayers treated with LUF7244 alone displayed electrophysiological properties indistinguishable from those of untreated NRVM cultures. Prolonged exposure of NRVMs to LUF7244 or LUF7244 plus astemizole did not affect their viability, excitability, and contractility as assessed by molecular, immunological, and electrophysiological assays. Conclusions: Allosteric modulation of the Kv11.1 channel efficiently suppresses drug-induced ventricular arrhythmias in vitro by preventing potentially arrhythmogenic changes in action potential characteristics, raising the possibility to resume the clinical use of unintended Kv11.1 blockers via pharmacological combination therapy. [ABSTRACT FROM AUTHOR]

Radioligand binding assays on intact cells offer distinct advantages to those on membrane suspensions. Major pharmacological properties like drug affinity and binding kinetics are more physiologically relevant. Complex mechanisms can be studied with a wider choice of experimental approaches and so provide insights into induced-fit type binding, receptor internalisation and even into pharmacomicrokinetic phenomena like drug rebinding and partitioning into the membrane. Hence, intact cell binding constitutes a valuable addition to the pharmacologist's toolbox. [ABSTRACT FROM AUTHOR]

A simple methodology was developed to evaluate binding efficiency of antibiotic members of fluoroquinolones, namely ciprofloxacin, ofloxacin and enorfloxacin, complexed with 99m Tc, against Escherichia coli , Salmonella and Pseudomonas aeruginosa bacterial strains. Radioactivity in the pellet, tips supernatant and micro-centrifugation tubes was counted separately for 5 s in a sodium iodide well-counter with dedicated nuclear medicine software. The overall percentage activity of the live and killed bacteria was found in the range of 5–46% with different types of labelled quinolones and bacteria. Activity of the labelled enorfloxacin and ciprofloxacin indicated acceptable results for both live and killed E. coli and Ps. aeruginosa. However, ofloxacin was found to be moderate for all the live bacterial stains. This developed methodology has achieved more than 95% labelling efficiency of 99m Tc with derivatized quinolones and also the observed results indicated that these complexes may be used as an infection specific imaging agents. [ABSTRACT FROM AUTHOR]

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is becoming another “SARS-like” threat to the world. It has an extremely high death rate (∼50%) as there is no vaccine or efficient therapeutics. The identification of the structures of both the MERS-CoV receptor binding domain (RBD) and its complex with dipeptidyl peptidase 4 (DPP4), raises the hope of alleviating this currently severe situation. In this review, we examined the molecular basis of the RBD-receptor interaction to outline why/how could we use MERS-CoV RBD to develop vaccines and antiviral drugs. [ABSTRACT FROM AUTHOR]

Abstract: The Estrogen Receptor (ER) is an established predictive marker for the selection of adjuvant endocrine treatment in early breast cancer. During the 1990s Immunohistochemistry (IHC) replaced cytosol based assays for determination of ER status. This study examined the association between ER protein level determined by two different methods and ESR1 gene copy number. From 289 primary high-risk breast cancer patients, randomized in the Danish Breast Cancer Cooperative Group (DBCG) 77C trial, results from cytosolic ER levels were available from ligand binding assays. Archival tumor tissue was retrieved from 257 patients. ESR1/CEN-6 ratio was analyzed successfully by Fluorescence In Situ Hybridization (FISH) in 220 (86%) patients. ESR1 amplification (ESR1/CEN-6 ≥ 2.00) was observed in 23% of the patients and ESR1 deletion (ESR1/CEN-6 < 0.80) was observed in 32%. Further, we identified ESR1 gain (ratio ESR1/CEN-6 from 1.30 to 1.99) in 19% of the patients. A positive correlation of ESR1 FISH with both ER-cytosol and ER IHC was found (p < 0.0001). Amplification and gain of the ESR1 gene are associated with higher ER protein content measured by ligand binding assay and a more intense nuclear staining by IHC compared to tumors with normal ESR1 gene status. Major variations in ER measured by ligand binding assay and IHC are observed within all ESR1 copy number subgroups and other mechanisms than gene copy number seem to contribute to the ER protein content in the tumors. [Copyright &y& Elsevier]

Abstract: Objective: Both the 2, 6 linkage and its topology on target cells are critical for the recognition by human influenza virus. The binding preference of avian flu virus H5N1 HA to the 2, 3-linked sialylated glycans is considered the major factor limiting its efficient infection and transmission in humans. To monitor potential adaptation of H5N1 virus in human population, the surveillance of receptor-binding specificity was undertaken in China. Methods: The binding specificity of 32 human H5N1 virus strains isolated from 2003 to 2009 was tested by 2, 3-specific sialidase-treated chicken red blood cell (CRBC) agglutination assay and a solid-phase direct binding assay with synthetic sialylglycopolymers. Results: Dual binding preference to 2, 3 and 2, 6-glycans were found in two strains: A/Guangdong/1/06 (A/GD/1/06) and A/Guangxi/1/08 (A/GX/1/08). Though minor effect of short-2, 6-binding was detected in A/GX/1/08 at a low virus titer, both showed high affinity to the oligosaccharide at a high load. Notably both are of the long-2, 6-recognition, with the same topology as that of human H1N1 and H3N2 viruses. Conclusion: The findings suggest that human H5N1 virus in China likely acquired the potential human-adaptation ability. Further research and surveillance on receptor-binding specificity of H5N1 viruses are required. [Copyright &y& Elsevier]

The number of N-linked glycosylation sites in the globular head of hemagglutinin (HA) has increased during evolution of H3N2 human influenza A virus. Here natural selection operating on the gains of N-linked glycosylation sites was examined by using the single-site analysis and the single-substitution analysis. In the single-site analysis, positive selection was not inferred at the amino acid sites where the substitutions generating N-linked glycosylation sites were observed, but was detected at antigenic sites. In contrast, in the single-substitution analysis, positive selection was detected for the amino acid substitutions generating N-linked glycosylation sites. The single-site analysis and the single-substitution analysis appeared to be suitable for detecting recurrent and episodic natural selection, respectively. The gains of N-linked glycosylation sites were likely to be positively selected for the function of shielding antigenic sites from immune responses. At the antigenic sites, positive selection appeared to have operated not only on the radical substitution but also on the conservative substitution in terms of the charge of amino acids, suggesting that the antigenic drift is not a by-product of the evolution of receptor binding avidity in HA of human H3N2 virus. [ABSTRACT FROM AUTHOR]

Abstract: A robust, selective and highly sensitive chemiluminescent (CL) platform for protein assay was presented in this paper. This novel CL approach utilized rolling circle amplification (RCA) as a signal enhancement technique and the 96-well plate as the immobilization and separation carrier. Typically, the antibody immobilized on the surface of 96-well plate was sandwiched with the protein target and the aptamer–primer sequence. This aptamer–primer sequence was then employed as the primer of RCA. Based on this design, a number of the biotinylated probes and streptavidin–horseradish peroxidase (SA–HRP) were captured on the plate, and the CL signal was amplified. In summary, our results demonstrated a robust biosensor with a detection limit of 10fM that is easy to be established and utilized, and devoid of light source. Therefore, this new technique will broaden the perspective for future development of DNA-based biosensors for the detection of other protein biomarkers related to clinical diseases, by taking advantages of high sensitivity and selectivity. [Copyright &y& Elsevier]

Envelope virus replication begins with receptor binding, followed by fusion of the viral envelope with the cell membrane. The binding and fusion steps are usually mediated by envelope proteins. The ability of envelope proteins of a particular virus to bind and fuse with target cells defines the host range of the virus, known as ‘viral tropism’. The mechanism(s) of fusion by the viral envelope is largely categorized as either pH-dependent or pH-independent. By redirecting the binding specificities of envelope proteins to desired target molecules while maintaining fusion activity, it is possible to redirect the tropisms of virus and viral vectors, enabling specific killing and/or transduction of desired cells in vivo. Recently, a lipid, phosphatidylserine, was also shown to mediate binding of virus, which affects the tropisms of viruses and viral vectors. [Copyright &y& Elsevier]

The effect of monovalent cations on trimeric G protein Gi1α was measured at equimolar concentration of chloride anion in pertussis-toxin (PTX)-treated HEK293 cells stably expressing PTX-insensitive DOR-Gi1α (Cys351 -Ile351 ) fusion protein by high-affinity [35S]GTPγS binding assay. The high basal level of binding was detected in absence of DOR agonist and monovalent ions and this high level was inhibited with the order of: Na+ > K+ Li+. The first significant inhibition was detected at 1 mM NaCl. The inhibition by monovalent ions was reversed by increasing concentrations of DOR agonist DADLE. The maximum DADLE response was also highest for sodium and decreased in the order of: Na+ > K+ ≈ Li+. Our data indicate i) an inherently high activity of trimeric G protein Gi1α when expressed within DOR-Gi1α fusion protein and determined in the absence of monovalent cations, ii) preferential sensitivity of DOR-Gi1α to sodium as far as maximum of agonist response is involved. [ABSTRACT FROM AUTHOR]

Abstract: The purpose of this study was to investigate whether the inflammation of rat dental pulp induces the muscarinic acetylcholine receptor (mAChR) constitutive receptor activity. Pulpitis was induced with bacterial lipolysaccharide in rat incisors dental pulp. Saturation assay with [3H]-quinuclidinyl benzilate ([3H] QNB), competitive binding with different mAChR antagonist subtypes, and nitric oxide synthase (NOS) activity were performed. A drastic change in expression and response to mAChR subtypes was observed in pulpitis. Inflamed pulp expressed high number of M3 mAChR of high affinity, whereas the M1 mAChR is the main subtype displayed in normal pulp. Consistent with the identification of the affinity constant (Ki) of M3 and Ki of M1 in both pulpitis and in normal pulps are the differences in the subtype functionality of these cells. In pulpitis, pilocarpine (1 × 10−11 mol/L to 5 × 10−9 mol/L) exerted an inhibitory action on NOS activity that was blocked by J 104129 fumarate (highest selective affinity to M3 mAChR). In normal pulps, pilocarpine (1 × 10−11 mol/L to 5 × 10−9 mol/L) has no effect. NOS basal activity was 5.9 times as high in pulpitis as in the normal pulp as a result of the activation of inducible NOS. The irreversible pulpitis could induce a mAChR alteration, increasing the high-affinity receptor density and transduction-coupling efficiency of inducible NOS activity, leading to a spontaneously active conformation of the receptor. Pilocarpine acting as an inverse agonist might be useful therapeutically to prevent necrosis and subsequent loss of dental pulp. [ABSTRACT FROM AUTHOR]

The article discusses a study seeking to characterize Canine parvovirus 2 (CPV-2) in Greece. CPV-2 is an agent that causes hemorrhagic enteritis in dogs. Standard polymerase chain reaction (PCR) was used to detect CPV-2 while minor groove binder prove assays were used to identify genetic variants. The study found that CPV-2a strains are the most common variant in the country.

Abstract: The mechanisms by which proteins adsorb to surfaces of biomaterials have long been of interest. The present work started with the premise that small/hard and large/soft proteins will yield different sets of normalized frequency shift and dissipation signals when studied with a quartz crystal microbalance. The aim was to evaluate the usefulness of these raw data to study protein competition using protein incubations in sequence and from mixtures of albumin (BSA) and gamma-globulin (BGG) at various ratios. Increasing the concentration of BSA decreases the adsorption of subsequently incubated BGG. For BSA/BGG mixtures the dissipation is similar for all logarithmic molar ratios BGG/BSA below 1 but soon decreases when the molar ratio of BSA/BGG (and opposite for the normalized frequency shift) is above 1, indicating preferential binding of BGG. Modelling indicated that differences in the film shear modulus and viscosity depend more on the properties of the self-assembling mono-layers (SAMs) than on the proteins. Films high in BSA tentatively differ in film shear modulus and viscosity from that of films high in BGG but only on the hydrophobic surfaces. The results were encouraging as the raw data were deemed to be able to point at protein adsorption competition. [Copyright &y& Elsevier]

Abstract: Background: Human growth hormone (hGH) is a complex mixture of molecular isoforms. Gaps in our knowledge exist regarding the structures and biological significances of the uncharacterized hGH molecular variants. Mercaptoethanol-resistant 45-kDa human growth hormone (MER-45kDa hGH) is an extraordinarily stable disulfide-linked hGH homodimer whose biological significance is unknown. Objectives: To elucidate the pharmacokinetic abilities of dimeric MER-45-kDa hGH to bind to GH and prolactin (PRL) receptors and to elucidate its abilities to stimulate cell proliferation in lactogen-induced and somatogen-induced in vitro cell proliferation bioassays. Design: The binding of MER-45-kDa hGH to GH and PRL receptors was tested in radioreceptor assays (RRAs). Competitive displacements of [125I]-bovine GH from bovine liver membranes, [125I]-ovine PRL from lactating rabbit mammary gland membranes and [125I]-hGH from human IM-9 lymphocytes by unlabelled GHs, PRLs or dimeric MER-45-kDa hGH were evaluated. The abilities of dimeric MER-45-kDa hGH to stimulate proliferation of lactogen-responsive Nb2 lymphoma cells and to stimulate proliferation of somatogen-responsive T47-D human breast cancer cells were assessed by incubation of cells with GHs or PRLs and subsequently measuring growth using the MTS cell proliferation assay. Results: Dimeric MER-45-kDa hGH, compared to monomeric hGH, had reduced binding affinities to both GH and prolactin receptors. In a bovine liver GH radioreceptor assay its ED50 (197.5pM) was 40.8% that of monomeric hGH. In a human IM-9 lymphocyte hGH RRA its ED50 (2.96nM) was 26.2% that of monomeric hGH. In a lactating rabbit mammary gland prolactin RRA its ED50 (3.56nM) was 16.8% that of a monomeric hGH. Dimeric MER-45-kDa hGH, compared to monomeric hGH, had a diminished capacity to stimulate proliferation of cells in vitro. In a dose–response relationship assessing proliferation of Nb2 lymphoma cells its ED50 (191pM) was 18.0% that of monomeric hGH. While monomeric hGH stimulated a 2.2-fold proliferation of T47-D human breast cancer cells above vehicle control, dimeric MER-45-kDa hGH was unable to stimulate the cells to proliferate and slightly inhibited their proliferation to 77.6% that of control. Conclusions: The topological arrangement of monomeric hGHs to form an unusually stable disulfide-linked dimer markedly diminishes hGH''s binding affinities to both GH and PRL receptors and also drastically attenuates its ability to stimulate proliferation of cells in vitro. [Copyright &y& Elsevier]

The article presents a study on a components of a real-time Sumo-Binding assay for the analysis of the Small Ubiquitin-realated Modifier-SUMO-interacting motifs (SUMO-SIM) protein interaction network. It mentions that the SUMO-Binding can be applied to the identification of SIMs and to screening for the identification of novel SUMO-binding proteins. It also investigates the SIMs in SETDB1 and MCAF1 and MCAF1 to gain insight into the assembly of SETDB1-MCAF1-mediated gene silencing.

Purpose: Although α1L-adrenoceptor is recognized as a target of α1 antagonist therapy for benign prostatic hyperplasia, the most common techniques, such as immunohistochemistry and in situ hybridization, are not applicable to examine α1L-AR vs α1A-AR tissue distribution because α1L-AR is now considered another phenotype sharing the α1A-AR gene and protein molecule. We labeled the α1A and α1L-adrenoceptor selective antagonist silodosin (Kissei Pharmaceutical, Matsumoto, Japan) with the fluorophore Alexa Fluor® 488 (Alexa-488-silodosin) to visualize α1L-AR expression. Materials and Methods: Radioligand binding and functional bioassay experiments were done to assess α1-AR expression in Chinese hamster ovary cells and human prostate tissues. Confocal imaging was subsequently performed. Results: Although Alexa-488-silodosin had about 10 times lower affinity for all α1-AR subtypes than silodosin in binding and functional studies, it had high selectivity to α1A and α1L-ARs. Confocal imaging revealed clear localization of fluorescence on the membrane of Chinese hamster ovary cells expressing α1A-AR but not α1B-and α1D-ARs, and in the muscle layer of the human prostate. The fluorescent signal in Chinese hamster ovary cells disappeared in the presence of 3 nM prazosin but fluorescence was observed in the human prostate even in the presence of 100 nM prazosin. Conclusions: Alexa-488-silodosin is a powerful fluorescent probe with high selectivity to α1A and α1L-ARs. Thus, Alexa-488-silodosin successfully visualizes the site of α1L-ARs in the muscle layer of the human prostate without losing its distinct pharmacological profile. [Copyright &y& Elsevier]

The article discusses the study which focuses on impact of four assay on FeLIX T-lymphotropic dependent feline leukemia virus (FeLV-T) induces immunodeficiency in cats. In the study, it revealed that FeLV-T is fused with defective and cofactor for infection enveloped glycoprotein of an endogenous FeLV and mediated infection of binding a phosphate transporter established with a feline sarcoma-positive leukemia-negative cell line of gene expression. In addition, FeLV-T was infected with AH/FeLIX cells and induced severe phosphate transporter cells used for FeLIX dependent mutants in FeLV infected cats.

The article discusses the result of a study which analyzed blood serum collected from five nocturnal, neotropical bats using a competitive protein binding assay (CPBA) to determine concentrations of 25-hydroxyvitamin D [25(OH)D]. Free-ranging neotropical cave-roosting bats were captured with mist nets, harp traps and hand nets and their whole blood taken by venipuncture. Values for the serum were tested for normality using Kolmogorov-Smirnov test. Results showed that low circulating 25(OH)D values were observed in the three plant-visiting species, Artibeus jamaicensis, Brachyphylla cavernarum and Monophyllus redmani and suggests that New-World, plant-visiting bats possess an independent mechanism of calcium absorption that is independent of vitamin D.

Abstract: Conventionally, insulin is believed to induce a metabolic response, and IGF-I a mitogenic/differentiation response in vivo. However, several studies indicate that the roles of insulin and IGF-I may not be that easy to separate. In this study, insulin and IGF-I specificity in terms of gene regulation was investigated in primary human skeletal muscle cells before and after differentiation. Cell cultures were treated with 100nM insulin, IGF-I or nothing for 4h, and gene expression was subsequently determined using the Affymetrix microarray platform. Insulin and IGF-I receptor levels were determined by qRT-PCR and by radioligand binding assays. In primary myoblasts, insulin did not have any significant effect on gene expression, whereas IGF-I regulated 229 genes. In primary myotubes, insulin regulated 105 genes, whereas IGF-I regulated 697 genes. Additionally, 99 genes were found to be differentially regulated by insulin and IGF-I in a direct comparison. The majority of these genes were specifically regulated by IGF-I, 16 genes were regulated by both ligands, and no genes were regulated by only insulin. The microarray results correlated with low levels of insulin receptors compared to IGF-I receptors as determined by radioligand binding assays. In the myotubes, we did not identify any ligand specificity in terms of functional categories. The major difference between the two ligands was their respective potencies in gene regulation, which was higher for IGF-I than for insulin. This was true for genes involved in both mitogenic and metabolic regulations. The data suggest that IGF-I is a more important metabolic regulator in skeletal muscle than previously estimated. [Copyright &y& Elsevier]

Abstract: The bioassay-guided purification of chloroform extracts of Kalimeris indica (Linn) Schultz-Bip led to the isolation of three diacylglycerols: 1-O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-hexadecanoylglycerol (1), 1-O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-hexadecanoyl-3-O-α-(6-sulfoquinovopyranosyl)glycerol (2), 1-O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-hexadecanoyl-3-O-[α-d-galactopyranosyl(1→6)-O-β-d-galactopyranosyl]glycerol (3). Their structures were established on the basis of spectral and chemical methods. The glyceroglycolipids 2 and 3 exhibited significant PAF receptor binding inhibitory activities. Our studies have identified diacylglycolipids as a novel class of potent PAF antagonist. [Copyright &y& Elsevier]

Abstract: Prior research reported decreased oxytocin levels in specific brain regions correlated with disruptions in maternal care following gestational cocaine treatment in rats. Similarly, prenatal exposure to cocaine impaired subsequent maternal behavior in adulthood, but behavioral alterations were not associated with decreases in oxytocin levels in the same brain regions as were found in their cocaine-treated rat dams. To determine if other aspects of the oxytocin system are disrupted by cocaine treatment or prenatal exposure to cocaine during critical time points associated with maternal care, oxytocin mRNA transcription and receptor binding were examined on postpartum day two in relevant brain regions following gestational treatment with, or prenatal exposure to, either cocaine or saline. We hypothesized that oxytocin mRNA levels and receptor binding would be differentially affected by cocaine in the early postpartum period of dams and their offspring. Our findings indicate that gestational cocaine treatment resulted in significant increases in oxytocin mRNA levels in only the paraventricular nucleus of cocaine-treated dams, with almost significant increases in both generations in the supraoptic nucleus, but no significant effects of cocaine on receptor binding in either generation of dams. These findings indicate that in addition to oxytocin levels, cocaine treatment or prenatal exposure primarily affects oxytocin mRNA synthesis, with little effect on receptor binding in specific brain regions associated with maternal behavior in the early postpartum period of the rat. [Copyright &y& Elsevier]

Purpose: Silodosin (KMD-3213 or [(-)-1-(3-hydroxypropyl)-5-[(2R)-2-({2-[2-(2,2,2trifluoroethoxy)phenoxy]ethyl}amino)propyl]-2,3-dihydro-1H-indole-7-carboxamide]) (Kissei Pharmaceutical Co., Ltd., Matsumoto, Japan) is a selective antagonist for α-1A and α-1L adrenoceptors. Using this tritiated ligand the 2 α-1 adrenoceptors were examined in binding studies with intact tissue segments and membrane preparations of human prostate, and compared with functionally identified α-1 adrenoceptor. Materials and Methods: Binding assays with tissue segments and membrane preparations of human prostate samples were performed using [3H]-silodosin and binding affinities for various drugs were estimated. In functional experiments antagonist affinities were evaluated from the inhibitory potency against the contractile response to noradrenaline. Results: [3H]-silodosin bound to intact segments and membrane preparations of human prostate with subnanomolar affinity. [3H]-silodosin binding sites in intact segments were divided into 2 distinct components with different affinities for prazosin and RS-17053 (N-[2(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α, α-dimethyl1H-indole-3-ethanamine hydrochloride) (Research Biochemicals International, Natick, Massachusetts), while binding in membrane preparations showed single high affinity for these drugs. [3H]-silodosin binding sites also showed high affinity for silodosin and tamsulosin but low sensitivity to BMY 7378 (8-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspiro(4.5)decane-7,9-dione) (Research Biochemicals International) in intact segments and in membrane preparations. In functional experiments silodosin and tamsulosin potently inhibited the contractile response to noradrenaline but prazosin, RS-17053 and BMY 7378 showed low antagonistic affinity. Conclusions: The current binding studies in human prostate samples clearly show that α-1L and α-1A adrenoceptors coexist as pharmacologically distinct entities in intact tissues but not in crude membrane preparations. Also, α-1 adrenoceptors involved in the contractile response to noradrenaline are the α-1L subtype. [Copyright &y& Elsevier]

Abstract: Neuroimaging can be used to inform about a large range of responses to pharmacological challenges. In particular, it is the emergence of techniques available for the in vivo assessment of drug effects that has generated the most interest. Of these, functional magnetic resonance imaging is fast developing in allowing the assessment of the effect of pharmacological agents with good temporal and spatial resolution. However, an array of alternative techniques remains viable for assessment of drug action within the brain, e.g. the use of radioligands in receptor mapping and drug occupancy imaging methods, as well as measurement of endogenous neurotransmitter release. [Copyright &y& Elsevier]

PURPOSE: Previous studies have shown that serum levels of the degradation products of cytokeratins could be used as surrogate markers in the diagnosis and followup of patients with solid tumors, including tumors of the bladder. MATERIALS AND METHODS: The soluble cytokeratin 19 fragment CYFRA 21–1 was measured by solid phase radioimmunoassay in the serum of 142 patients with invasive transitional cell cancer of the bladder. Of the patients 56 had clinical stage I to III locally confined disease (T1–4aN0M0) and 86 had stage IV metastatic disease with lymph node and/or distant metastases. A control group consisted of 33 healthy volunteers. In a subgroup of 49 patients with metastatic disease receiving combined platinum based chemotherapy serum CYFRA 21–1 was determined prior to the initiation of therapy and after the documentation of response. RESULTS: Abnormal CYFRA 21–1 was observed in 7% of patients with locally invasive disease and in 66% of those with metastatic disease (p <0.0001). There was no correlation of CYFRA 21–1 with tumor differentiation. Patients with abnormal CYFRA 21–1 showed statistically significant worse median overall survival. Moreover, in the subgroup of patients with metastatic disease receiving chemotherapy CYFRA 21–1 levels correlated with the response to treatment. CONCLUSIONS: Patients with transitional cell cancer of the bladder with evidence of distant metastases showed a significant increase in serum CYFRA 21–1. During chemotherapy CYFRA 21–1 appears to be a potentially sensitive and useful indicator for monitoring treatment response. [ABSTRACT FROM AUTHOR]

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Abstract: Gene knockout approaches have helped to better understand the functions of the different Y receptors. However, some results obtained from these knockout mice are unexpected and differ from the results of pharmacological intervention experiments. One possible explanation for this is that germ-line gene deletion of a particular Y receptor can influence expression and function of the remaining Y receptors. Here we show that such compensation in mRNA and protein expression does occur in Y receptor single, double and triple knockout models. Radio-ligand binding experiments using [125I]-PYY revealed significant up- and down-regulation of remaining Y receptor binding sites in various Y receptor knockout models compared to results from control mice employing Y receptor preferring agonist or antagonists for displacement of the radio-ligand. The most obvious change can be seen in the hippocampus of Y1 knockout mice, where the level of the remaining Y receptors is strongly down-regulated. In Y2 knockout mice no such trend can be seen, however, the expression pattern is significantly changed with a strong up-regulation of [125I]-PYY specific binding in the dentate gyrus. Interestingly, this pattern was also seen in Y1Y2Y4 triple knockout mice. Y5 receptor mRNA was approximately 20% higher in the hippocampus and dentate gyrus in the triple knockout mice compared to wild-type controls, while Y6 mRNA expression could not be detected. However, competition binding experiments in Y1Y2Y4 triple knockout mice with the Y5 receptor preferring ligands [Leu31, Pro34] NPY and [A31, Aib32] NPY were able to replace only approximately 50% of [125I]-PYY binding in the dentate gyrus suggesting the existence of further yet unidentified Y receptor(s). [Copyright &y& Elsevier]

Despite strong homology between subunits of AMPA-type glutamate receptors, heteromeric assemblies in different brain regions vary in subunit composition and functional properties. We have previously shown that charge substitution at K445 decreases the apparent potency of AMPA for GluR-A receptors, although an analogous mutation is a low conductance B subunit of AB heteromers did not diminish AMPA potency. To examine the subunit selectivity of K445, we now measure the apparent potency of AMPA and glutamate at recombinant homomeric receptors containing the high-conductance, unedited (586Q) GluR-B subunit (GluR-B(Q)) using two-electrode voltage-clamp. We find the decrease in apparent potency of AMPA is recapitulated for homomeric GluR-B(Q)K449E receptors, and is rescued by coexpression with wild-type GluR-A. These data are consistent with the hypothesis that AMPAR subunit composition is critical to agonist-dependent receptor activation, and suggest that GluR-A plays a dominant role in mediating receptor activation by AMPA. [ABSTRACT FROM AUTHOR]

Background: Recently, an increased incidence of allergic diseases has been associated with vitamin D deficiency. We demonstrated previously that calcitriol, the active form of vitamin D, inhibits ε germline transcription, a prerequisite for IgE production. However, the underlying mechanisms remain unexplored. Objective: We sought to investigate whether the ε germline gene promoter (Iε) represents a primary vitamin D receptor (VDR) target. Therefore we investigated VDR binding to Iε, analyzed VDR-complex composition in more detail, and delineated its functional consequences. Methods: The VDR binding to Iε in human B cells, the composition of the VDR-recruited complex, and the acetylation pattern were investigated by means of chromatin immunoprecipitation. The calcitriol-mediated action on Iε was analyzed by using a reporter gene assay. Results: We demonstrate that Iε is a possible VDR target. Calcitriol-activated VDR binds together with retinoid X receptor α to the Iε region. The heterodimer interacts with silencing mediator for retinoid and thyroid hormone receptors, which recruits histone deacetylase (HDAC) 1 and HDAC3. The inhibition of silencing mediator for retinoid and thyroid hormone receptors or HDACs reversed the site-specific deacetylation of histones 3 and 4 and the calcitriol-driven inhibition of the ε germline transcription. The VDR-complex transrepressive actions on Iε were confirmed in a reporter assay. Conclusion: We show here that inhibition of IgE production by calcitriol is mediated by its transrepressive activity through the VDR-corepressor complex affecting chromatin compacting around the Iε region. Our findings shed new light on mechanisms of VDR transrepression and understanding of IgE regulation. [Copyright &y& Elsevier]

Background: Specificities for carbohydrate IgG antibodies, thought to be predominantly of the IgG2 subclass, have never been broadly examined in healthy human subjects. Objective: To examine commercial intravenous immunoglobulin (IVIG) preparations for their ability to recognize a wide range of glycans and to determine the contribution of IgG2 to the binding pattern observed. Methods: We used a glycan microarray to evaluate IVIG preparations and a control mix of similar proportions of human myeloma IgG1 and IgG2 for binding to 377 glycans, courtesy of the Consortium for Functional Glycomics Core H. Glycans recognized were categorized using public databases for their likely cellular sources. IgG2 was depleted from IVIG by using immunoaffinity chromatography, and depletion was confirmed by using nephelometry and surface plasmon resonance. Results: Nearly half of the glycans bound IgG. Some of the glycans with the greatest antibody binding can be found in structures of human pathogenic bacteria (eg, Streptococcus pneumoniae, Mycobacterium tuberculosis, Vibrio cholera) and nonpathogenic bacteria, including LPS and lipoteichoic acid, capsular polysaccharides, and exopolysaccharides. Surprisingly, depletion of IgG2 had only a modest effect on anticarbohydrate recognition patterns compared with the starting IVIG preparation. Little to no binding activity was detected to human endogenous glycans, including tumor-associated antigens. Conclusions: This novel, comprehensive analysis provides evidence that IVIG contains a much wider range than previously appreciated of anticarbohydrate IgG antibodies, including those recognizing both pathogenic and non–pathogen-associated prokaryotic glycans. [Copyright &y& Elsevier]