Receptor Assay Research Articles

ODP279 A Case of Nephrogenic Diabetes Insipidus Diagnosed at an Advanced Age in a Female Patient with an AVPR2 Gene Mutation and Skewed×Inactivation

Abstract Background Arginine vasopressin receptor 2 (AVPR2) gene mutationsare the most common cause of congenital nephrogenic diabetes insipidus (CNDI), and they are inherited in an X-linked recessive manner. Women are generally asymptomatic or mildly affected, but atypically severe cases have been reported. Skewed X inactivation, in which the X chromosome is unevenly active, is a cause of this atypical severe case.×chromosome inactivation is a developmental compensation mechanism that results in equal doses of X-linked genes between XX females and XY males and has been reported to be accentuated by aging. Clinical Case A 69-year-old woman had no family history of diabetes insipidus. Immediately after undergoing a craniotomy for subarachnoid hemorrhage in July, she showed marked polyuria of approximately 8000 mL/day and hypernatremia of 161 mEq/L. The use of desmopressin (15mcg/kg/day) did not improve herpolyuria. She was referred to our department in September for further investigation and treatment. Her urine osmolality remained hypotonic at approximately 60–80 mOsm/kg/H 2 O (50–1300 mOsm/kg/H 2 O) according to a water restriction test. Her urine osmolality increased slightly to 106 mOsm/kg/H 2 O in a desmopressin test. These results indicated that desmopressin was ineffective, and the patient was diagnosed with nephrogenic diabetes. Her urine output decreased to approximately 2500 mL/day with trichlormethiazide treatment. There was no significant anterior pituitary hypofunction. Genetic examination revealed a heterozygous mutation of the AVPR2 gene (c.656T>G, p. Leu219Arg) in her X chromosome, and her X chromosome activity ratio was 79: 21 in a human androgen receptor assay, indicating skewed X inactivation. Conclusion We observed a case of CNDI in an elderly woman with no family history of the disease. In this case, a combination of the skewed X inactivation, presumably accentuated by aging, and the onset of subarachnoid hemorrhage, reduced her drinking water and led to the diagnosis of CNDI.

Renal X-inactivation in female individuals with X-linked Alport syndrome primarily determined by age

X-linked Alport syndrome (AS) caused by hemizygous disease-causing variants in COL4A5 primarily affects males. Females with a heterozygous state show a diverse phenotypic spectrum ranging from microscopic hematuria to end-stage kidney disease (ESKD) and extrarenal manifestations. In other X-linked diseases, skewed X-inactivation leads to preferential silencing of one X-chromosome and thus can determine the phenotype in females. We aimed to show a correlation between X-inactivation in blood and urine-derived renal cells and clinical phenotype of females with a heterozygous disease-causing variant in COL4A5 compared to healthy controls. A total of 56 females with a heterozygous disease-causing COL4A5 variant and a mean age of 31.6 ± 18.3 SD years were included in this study. A total of 94% had hematuria, 62% proteinuria >200 mg/day, yet only 7% had decreased eGFR. Using human androgen receptor assay X-inactivation was examined in blood cells of all 56 individuals, in urine-derived cells of 27 of these individuals and in all healthy controls. X-inactivation did not correlate with age of first manifestation, proteinuria or eGFR neither in blood, nor in urine. The degree of X-inactivation showed a moderate association with age, especially in urine-derived cells of the patient cohort (rho = 0.403, p = 0.037). Determination of X-inactivation allelity revealed a shift of X-inactivation toward the COL4A5 variant bearing allele. This is the first study examining X-inactivation of urine-derived cells from female individuals with AS. A correlation between phenotype and X-inactivation could not be observed suspecting other genetic modifiers shaping the phenotype in female individuals with AS. The association of X-inactivation with age in urine-derived cells suggests an escape-mechanism inactivating the COL4A5 variant carrying allele in female individuals with AS.

Detection of Bitterness in Vitamins Is Mediated by the Activation of Bitter Taste Receptors.

Vitamins are known to generate bitterness, which may contribute to an off-taste or aftertaste for some nutritional supplements. This negative sensation can lead to a reduction in their consumption. Little is known about the bitter taste threshold and taste sensing system for the bitter taste detection of vitamins. To better understand the mechanisms involved in bitterness perception, we combined taste receptor functional assays and sensory analysis. In humans, bitter taste detection is mediated by 25 G-protein-coupled receptors belonging to the TAS2R family. First, we studied the bitterness of thirteen vitamins using a cellular-based functional taste receptor assay. We found four vitamins that can stimulate one or more TAS2Rs. For each positive molecule–receptor combination, we tested seven increasing concentrations to determine the half-maximal effective concentration (EC50) and the cellular bitter taste threshold. Second, we measured the bitter taste detection threshold for four vitamins that exhibit a strong bitter taste using a combination of ascending series and sensory difference tests. A combination of sensory and biological data can provide useful results that explain the perception of vitamin bitterness and its real contribution to the off-taste of nutritional supplements.

Residues in the 1st Transmembrane-Spanning Helix Are Important for GABAAρ Receptor Function.

GABAAρ receptors are a subfamily of the GABAA receptor family of pentameric ligand-gated ion channels (pLGICs). Each subunit has a common structure, including a transmembrane domain of four α-helices (M1–M4). The aim of this study was to identify important M1 residues in the GABAAρ receptor (GABAAρR), using mutagenesis and functional assays combined with bioinformatic approaches. Alanine substitution of 12 of the 23 M1 residues yielded receptors with altered functional parameters, indicating these residues contribute to GABAAρR function. Further mutations reveal the properties that are important for function in critical residues, and, using a GABAAρR homology model, we suggest amino acid interactions that could be important. Phylogenetic analysis comparing GABAAR and other pLGICs subunits reveals most M1 residue properties linked to GABAAρR function are ancestrally ancient, but some are more recent acquisitions. Multiple sequence alignment of M1 residues across GABAAR subunits reveal three residues are well conserved except in GABAAR α subunits. Substitution of ρ1 subunit residues to their α1 subunit equivalents showed one alters functional parameters. Overall, the data provide a comprehensive picture of M1 residues that contribute to GABAAρR function, and illustrate how they might do so.

Structural basis for recognition of N-formyl peptides as pathogen-associated molecular patterns

The formyl peptide receptor 1 (FPR1) is primarily responsible for detection of short peptides bearing N-formylated methionine (fMet) that are characteristic of protein synthesis in bacteria and mitochondria. As a result, FPR1 is critical to phagocyte migration and activation in bacterial infection, tissue injury and inflammation. How FPR1 distinguishes between formyl peptides and non-formyl peptides remains elusive. Here we report cryo-EM structures of human FPR1-Gi protein complex bound to S. aureus-derived peptide fMet-Ile-Phe-Leu (fMIFL) and E. coli-derived peptide fMet-Leu-Phe (fMLF). Both structures of FPR1 adopt an active conformation and exhibit a binding pocket containing the R2015.38XXXR2055.42 (RGIIR) motif for formyl group interaction and receptor activation. This motif works together with D1063.33 for hydrogen bond formation with the N-formyl group and with fMet, a model supported by MD simulation and functional assays of mutant receptors with key residues for recognition substituted by alanine. The cryo-EM model of agonist-bound FPR1 provides a structural basis for recognition of bacteria-derived chemotactic peptides with potential applications in developing FPR1-targeting agents.

Identification of nonmonotonic concentration-responses in Tox21 high-throughput screening estrogen receptor assays

Environmental endocrine-disrupting chemicals (EDCs) interfere with the metabolism and actions of endogenous hormones. It has been well documented in numerous in vivo and in vitro studies that EDCs can exhibit nonmonotonic dose response (NMDR) behaviors. Not conforming to the conventional linear or linear-no-threshold response paradigm, these NMDR relationships pose practical challenges to the risk assessment of EDCs. In the meantime, the endocrine signaling pathways and biological mechanisms underpinning NMDR remain incompletely understood. The US Tox21 program has conducted in vitro cell-based high-throughput screening assays for estrogen receptors (ER), androgen receptors, and other nuclear receptors, and screened the 10 K-compound library for potential endocrine activities. Using 15 concentrations across several orders of magnitude of concentration range and run in both agonist and antagonist modes, these Tox21 assay datasets contain valuable quantitative information that can be explored to evaluate the nonlinear effects of EDCs and may infer potential mechanisms. In this study we analyzed the concentration-response curves (CRCs) in all 8 Tox21 ERα and ERβ assays by developing clustering and classification algorithms customized to the datasets to identify various shapes of CRCs. After excluding NMDR curves likely caused by cytotoxicity, luciferase inhibition, or autofluorescence, hundreds of compounds were identified to exhibit Bell or U-shaped CRCs. Bell-shaped CRCs are about 7 times more frequent than U-shaped ones in the Tox21 ER assays. Many compounds exhibit NMDR in at least one assay, and some EDCs well-known for their NMDRs in the literature were also identified, suggesting their nonmonotonic effects may originate at cellular levels involving transcriptional ER signaling. The developed computational methods for NMDR identification in ER assays can be adapted and applied to other high-throughput bioassays.

Arabidopsis flowering integrator SOC1 transcriptionally regulates autophagy in response to long-term carbon starvation.

Autophagy is a highly conserved, self-digestion process that is essential for plant adaptations to various environmental stresses. Although the core components of autophagy in plants have been well established, the molecular basis for its transcriptional regulation remains to be fully characterized. In this study, we demonstrate that SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), a MADS-box family transcription factor that determines flowering transition in Arabidopsis, functions as a transcriptional repressor of autophagy. EMSAs, ChIP-qPCR assays, and dual-luciferase receptor assays showed that SOC1 can bind to the promoters of ATG4b, ATG7, and ATG18c via the conserved CArG box. qRT-PCR analysis showed that the three ATG genes ATG4b, ATG7, and ATG18c were up-regulated in the soc1-2 mutant. In line with this, the mutant also displayed enhanced autophagy activity, as revealed by increased autophagosome formation and elevated autophagic flux compared with the wild type. More importantly, SOC1 negatively affected the tolerance of plants to long-term carbon starvation, and this process requires a functional autophagy pathway. Finally, we found that SOC1 was repressed upon carbon starvation at both the transcriptional and protein levels. Overall, our study not only uncovers an important transcriptional mechanism that contributes to the regulation of plant autophagy in response to nutrient starvation, but also highlights novel cellular functions of the flowering integrator SOC1.

Luminogenic HiBiT Peptide-Based NanoBRET Ligand Binding Assays for Melatonin Receptors.

The two human melatonin receptors MT1 and MT2, which belong to the G protein-coupled receptor (GPCR) family, are important drug targets with approved indications for circadian rhythm- and sleep-related disorders and major depression. Currently, most of the pharmacological studies were performed using [3H]melatonin and 2-[125I]iodomelatonin (2-[125I]-MLT) radioligands. Recently, NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) monitoring competitive binding between fluorescent tracers and unmodified test compounds has emerged as a sensitive, nonradioactive alternative for quantifying GPCR ligand engagement on the surface of living cells in equilibrium and real time. However, developing such assays for the two melatonin receptors depends on the availability of fluorescent tracers, which has been challenging predominantly owing to their narrow ligand entry channel and small ligand binding pocket. Here, we generated a set of melatonergic fluorescent tracers and used NanoBRET to evaluate their engagement with MT1 and MT2 receptors that are genetically fused to an N-terminal luminogenic HiBiT-peptide. We identified several nonselective and subtype-selective tracers. Among the selective tracers, PBI-8238 exhibited high nanomolar affinity to MT1, and PBI-8192 exhibited low nanomolar affinity to MT2. The pharmacological profiles of both tracers were in good agreement with those obtained with the current standard 2-[125I]-MLT radioligand. Molecular docking and mutagenesis studies suggested the binding mode of PBI-8192 in MT2 and its selectivity over MT1. In conclusion, we describe the development of the first nonradioactive, real-time binding assays for melatonin receptors expressed at the cell surface of living cells that are likely to accelerate drug discovery for melatonin receptors.

In vitro activity of a panel of per- and polyfluoroalkyl substances (PFAS), fatty acids, and pharmaceuticals in peroxisome proliferator-activated receptor (PPAR) alpha, PPAR gamma, and estrogen receptor assays

Data demonstrate numerous per- and polyfluoroalkyl substances (PFAS) activate peroxisome proliferator-activated receptor alpha (PPARα), however, additional work is needed to characterize PFAS activity on PPAR gamma (PPARγ) and other nuclear receptors. We utilized in vitro assays with either human or rat PPARα or PPARγ ligand binding domains to evaluate 16 PFAS (HFPO-DA, HFPO-DA-AS, NBP2, PFMOAA, PFHxA, PFOA, PFNA, PFDA, PFOS, PFBS, PFHxS, PFOSA, EtPFOSA, and 4:2, 6:2 and 8:2 FTOH), 3 endogenous fatty acids (oleic, linoleic, and octanoic), and 3 pharmaceuticals (WY14643, clofibrate, and the metabolite clofibric acid). We also tested chemicals for human estrogen receptor (hER) transcriptional activation. Nearly all compounds activated both PPARα and PPARγ in both human and rat ligand binding domain assays, except for the FTOH compounds and PFOSA. Receptor activation and relative potencies were evaluated based on effect concentration 20% (EC20), top percent of max fold induction (pmaxtop), and area under the curve (AUC). HFPO-DA and HFPO-DA-AS were the most potent (lowest EC20, highest pmaxtop and AUC) of all PFAS in rat and human PPARα assays, being slightly less potent than oleic and linoleic acid, while NBP2 was the most potent in rat and human PPARγ assays. Only PFHxS, 8:2 and 6:2 FTOH exhibited hER agonism >20% pmax. In vitro measures of human and rat PPARα and PPARγ activity did not correlate with oral doses or serum concentrations of PFAS that induced increases in male rat liver weight from the National Toxicology Program 28-d toxicity studies. Data indicate that both PPARα and PPARγ activation may be molecular initiating events that contribute to the in vivo effects observed for many PFAS.

EPEN-05. Adenosine receptor expression in paediatric ependymoma

PURPOSE: Paediatric ependymoma is associated with dismal outcomes. Whilst understanding of its underlying biology has advanced, there has been little progress in treatment and clinical outcomes. Acting through four G protein-coupled receptors (encoded by ADORA1, ADORA2A, ADORA2B and ADORA3), adenosine is a signalling molecule often present at high levels in tumours. Adenosine signalling can aid tumour proliferation and invasiveness via mechanisms including suppression of tumour-infiltrating immune cells. Adenosine receptors therefore represent a potential therapeutic target in paediatric ependymoma, however neither levels nor patterns of expression have been previously reported. We hypothesised that adenosine receptors would be expressed in paediatric ependymoma and that this expression would vary between molecular subgroups. METHODS: Three publicly available gene expression datasets were analysed for adenosine receptor expression using Kruskal-Wallis, Mann-Whitney U and chi-square tests. RNAscope assays for adenosine receptors and CD68 were then performed on ten full-face ependymoma FFPE sections from posterior fossa A (PFA1 and PFA2) tumours to understand patterns of expression within ependymomas with the highest levels of expression identified by the gene expression datasets. RESULTS: Statistically significant differences were identified between adenosine-related genes across ependymoma subgroups of differing anatomical origin (supratentorial ZFTA-positive versus posterior fossa A and B (PFA/PFB)), with median adenosine-related gene levels generally higher in the PFA subgroup. Particularly, ADORA1, 2A, 2B and 3 gene expression was higher in PFA tumours than other subgroups. Analysis of the ten cases demonstrated measurable expression of all four adenosine receptors by RNAscope and patterns in the distribution and relative levels of expression of the adenosine receptors across PFA1 and PFA2 tumours were described. CONCLUSION: Using two different techniques we demonstrated that adenosine receptors are expressed in paediatric ependymomas. There are significant differences in level of expression between tumour subgroups. Adenosine receptors therefore represent a potential therapeutic target which should be explored further.

823-P: The Long-Acting Dual Amylin and Calcitonin Receptor Agonist KBP Has Increased Efficacy on Weight Loss and Glucose Control Compared with Cagrilintide in Obese and Diabetic Rats

Long-acting dual amylin and calcitonin receptor agonists (DACRAs) are novel candidates for treatment of type 2 diabetes (T2D) and obesity due to their beneficial effects on both body weight, glucose control and insulin action. Cagrilintide, which is currently in clinical trials, has shown promising effects on weight loss. In this study we compared a new long-acting DACRA (KBP) to cagrilintide in pre-clinical models of obesity and T2D. In vitro potencies were assessed using receptor assays. In vivo efficacies were investigated head-to-head in high fat diet (HFD) fed obese and T2D (ZDF) rat models. In vitro data showed that both peptides active both the amylin and the calcitonin receptor, with KBP being more potent on both receptors. This was further confirmed in vivo by assessment of acute effects on food intake and CTX suppression. KBP (1.5, 4.5 and 13.5 nmol/kg) and cagrilintide (10, 30 and 100 nmol/kg) induced a potent and dose-dependent weight loss in HFD rats, with the highest dose of KBP being superior to cagrilintide. In diabetic ZDF rats, DACRA treatment improved glucose control and preserved plasma insulin compared to vehicle. Interestingly, despite similar levels of plasma insulin, KBP treatment was superior to cagrilintide in improving glucose control. This was further reflected in the HbA1c levels at study end, where KBP treatment resulted in significantly lower levels compared to cagrilintide. In summary, both DACRAs induced weight loss and improved glucose tolerance, insulin action as well as glucose control. However, KBP treatment results in superior efficacy on both weight loss and glucose control. These findings highlight KBP as a promising once-weekly agent for treatment of obesity and T2D. Disclosure A.T.Larsen: Employee; Nordic Bioscience. N.Sonne: None. K.Mohamed: None. E.Bredtoft: None. F.Andersen: None. M.A.Karsdal: Employee; Nordic Bioscience A/S, Nordic Bioscience A/S, Nordic Bioscience A/S. K.Henriksen: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S.

Bioanalytical and chemical-specific screening of contaminants of concern in three California (USA) watersheds

To broaden the scope of contaminants monitored in human-impacted riverine systems, water, sediment, and treated wastewater effluent were analyzed using receptor-based cell assays that provide an integrated response to chemicals based on their mode of biological activity. Samples were collected from three California (USA) watersheds with varying degrees of urbanization and discharge from municipal wastewater treatment plants (WWTPs). To complement cell assay results, samples were also analyzed for a suite of contaminants of emerging concern (CECs) using gas and liquid chromatography-mass spectrometry (GC- and LC-MS/MS). For most water and sediment samples, bioassay equivalent concentrations for estrogen and glucocorticoid receptor assays (ER- and GR-BEQs, respectively) were near or below reporting limits. Measured CEC concentrations compared to monitoring trigger values established by a science advisory panel indicated minimal to moderate concern in water but suggested that select pesticides (pyrethroids and fipronil) had accumulated to levels of greater concern in river sediments. Integrating robust, standardized bioanalytical tools such as the ER and GR assays utilized in this study into existing chemical-specific monitoring and assessment efforts will enhance future CEC monitoring efforts in impacted riverine systems and coastal watersheds.

Holistic approach to chemical and microbiological quality of aquatic ecosystems impacted by wastewater effluent discharges

Wastewater treatment plants (WWTPs) collect wastewater from various sources and use different treatment processes to reduce the load of pollutants in the environment. Since the removal of many chemical pollutants and bacteria by WWTPs is incomplete, they constitute a potential source of contaminants. The continuous release of contaminants through WWTP effluents can compromise the health of the aquatic ecosystems, even if they occur at very low concentrations. The main objective of this work was to characterize, over a period of four months, the treatment steps starting from income to the effluent and 5 km downstream to the receiving river. In this context, the efficiency removal of chemical pollutants (e.g. hormones and pharmaceuticals, including antibiotics) and bacteria was assessed in a WWTP case study by using a holistic approach. It embraces different chemical and biological-based methods, such as pharmaceutical analysis by HPLC-MSMS, growth rate inhibition in algae, ligand binding estrogen receptor assay, microbial community study by 16S and shotgun sequencing along with relative quantification of resistance genes by quantitative polymerase chain reaction. Although both, chemical and biological-based methods showed a significant reduction of the pollutant burden in effluent and surface waters compared to the influent of the WWTP, no complete removal of pollutants, pathogens and antibiotic resistance genes was observed.

Pharmacology of Kappa Opioid Receptors: Novel Assays and Ligands.

The present study investigated the in vitro pharmacology of the human kappa opioid receptor using multiple assays, including calcium mobilization in cells expressing chimeric G proteins, the dynamic mass redistribution (DMR) label-free assay, and a bioluminescence resonance energy transfer (BRET) assay that allows measurement of receptor interaction with G protein and β-arrestin 2. In all assays, dynorphin A, U-69,593, and [D-Pro10]dyn(1-11)-NH2 behaved as full agonists with the following rank order of potency [D-Pro10]dyn(1-11)-NH2 > dynorphin A ≥ U-69,593. [Dmt1,Tic2]dyn(1-11)-NH2 behaved as a moderate potency pure antagonist in the kappa-β-arrestin 2 interaction assay and as low efficacy partial agonist in the other assays. Norbinaltorphimine acted as a highly potent and pure antagonist in all assays except kappa-G protein interaction, where it displayed efficacy as an inverse agonist. The pharmacological actions of novel kappa ligands, namely the dynorphin A tetrameric derivative PWT2-Dyn A and the palmitoylated derivative Dyn A-palmitic, were also investigated. PWT2-Dyn A and Dyn A-palmitic mimicked dynorphin A effects in all assays showing similar maximal effects but 3–10 fold lower potency. In conclusion, in the present study, multiple in vitro assays for the kappa receptor have been set up and pharmacologically validated. In addition, PWT2-Dyn A and Dyn A-palmitic were characterized as potent full agonists; these compounds are worthy of further investigation in vivo for those conditions in which the activation of the kappa opioid receptor elicits beneficial effects e.g. pain and pruritus.

Putative Synthetic Cannabinoids MEPIRAPIM, 5F-BEPIRAPIM (NNL-2), and Their Analogues Are T-Type Calcium Channel (CaV3) Inhibitors.

Synthetic cannabinoid receptor agonists (SCRAs) are a large and growing class of new psychoactive substances (NPSs). Two recently identified compounds, MEPIRAPIM and 5F-BEPIRAPIM (NNL-2), have not been confirmed as agonists of either cannabinoid receptor subtype but share structural similarities with both SCRAs and a class of T-type calcium channel (CaV3) inhibitors under development as new treatments for epilepsy and pain. In this study, MEPIRAPIM and 5F-BEPIRAPIM and 10 systematic analogues were synthesized, analytically characterized, and pharmacologically evaluated using in vitro cannabinoid receptor and CaV3 assays. Several compounds showed micromolar affinities for CB1 and/or CB2, with several functioning as low potency agonists of CB1 and CB2 in a membrane potential assay. 5F-BEPIRAPIM and four other derivatives were identified as potential CaV3 inhibitors through a functional calcium flux assay (>70% inhibition), which was further confirmed using whole-cell patch-clamp electrophysiology. Additionally, MEPIRAPIM and 5F-BEPIRAPIM were evaluated in vivo using a cannabimimetic mouse model. Despite detections of MEPIRAPIM and 5F-BEPIRAPIM in the NPS market, only the highest MEPIRAPIM dose (30 mg/kg) elicited a mild hypothermic response in mice, with no hypothermia observed for 5F-BEPIRAPIM, suggesting minimal central CB1 receptor activity.

Preferential X Chromosome Inactivation as a Mechanism to Explain Female Preponderance in Myasthenia Gravis.

Myasthenia gravis (MG) is a neuromuscular autoimmune disease characterized by prevalence in young women (3:1). Several mechanisms proposed as explanations for gender bias, including skewed X chromosome inactivation (XCI) and dosage or sex hormones, are often involved in the development of autoimmunity. The skewed XCI pattern can lead to an unbalanced expression of some X-linked genes, as observed in several autoimmune disorders characterized by female predominance. No data are yet available regarding XCI and MG. We hypothesize that the preferential XCI pattern may contribute to the female bias observed in the onset of MG, especially among younger women. XCI analysis was performed on blood samples of 284 women between the ages of 20 and 82. XCI was tested using the Human Androgen Receptor Assay (HUMARA). XCI patterns were classified as random (XCI < 75%) and preferential (XCI ≥ 75%). In 121 informative patients, the frequency of skewed XCI patterns was 47%, significantly higher than in healthy controls (17%; p ≤ 0.00001). Interestingly, the phenomenon was observed mainly in younger patients (<45 years; p ≤ 0.00001). Furthermore, considering the XCI pattern and the other clinical characteristics of patients, no significant differences were found. In conclusion, we observed preferential XCI in MG female patients, suggesting its potential role in the aetiology of MG, as observed in other autoimmune diseases in women.

Alcohol exacerbated biochemical and biophysical alterations in liver mitochondrial membrane of diabetic male wistar rats – A possible amelioration by green tea

Alcohol exacerbated biochemical and biophysical alterations in liver mitochondrial membrane of diabetic male wistar rats – A possible amelioration by green tea

Myotropic Activities of Tick Pyrokinin Neuropeptides and Analog in Feeding Tissues of Hard Ticks (Ixodidae).

Neuropeptides regulate many important physiological processes in animals. The G protein-coupled receptors of corresponding small neuropeptide ligands are considered promising targets for controlling arthropod pests. Pyrokinins (PKs) are pleiotropic neuropeptides that, in some insect species, stimulate muscle contraction and modulate pheromone biosynthesis, embryonic diapause, and feeding behavior. However, their function remains unknown in ticks. In this study, we reported the myotropic activity of tick endogenous PKs and a PK agonist analog, PK-PEG8 (MS[PEG8]-YFTPRLa), on feeding tissues of two tick species representing the family Ixodidae lineages, namely, Prostriata (Ixodes scapularis) and Metastriata (Rhipicephalus sanguineus). First, we predicted the sequences of two periviscerokinins (PVK), one with a derived ending RNa and five PKs encoded by the CAPA peptide precursor from R. sanguineus and found the encoded PKs were identical to those of R. microplus identified previously. The pharynx-esophagus of both tick species responded with increased contractions to 10 μM of the endogenous PK as well as to PK-PEG8 but not to the scrambled PK peptide, as expected. A dose-dependent myotropic activity of the PK-PEG8 was found for both tick species, validating the analog activity previously found in the pyrokinin recombinant receptor assay. In agreement with the tissue activity elicited, we quantified the relative transcript abundance of R. sanguineus PK receptor in unfed female ticks and found it was the highest in the feeding tissues extracted from the capitulum and lowest in the reproductive tissue. This is the first report of the activity of pyrokinins in ticks. These findings strongly indicate the potential role of PKs in regulating tick blood feeding and therefore, making the tick PK receptor a potential target for interference.

Conjugates of Aminopenicillins with Proteins: Synthesis, Immunogenic Properties, and Binding to the \u03b2-Lactam Receptor and Antibodies

A new approach to aminopenicillin modification and conjugation with proteins was developed using di-N-hydroxysuccinimide esters of dicarboxylic acids as crosslinkers. Acylation of ampicillin (Amp) and amoxicillin (Amox) with di-N-hydroxysuccinimide esters of adipic or terephthalic acids was carried out in an organic solvent. Subsequent conjugation of the resulting aminopenicillin derivatives with proteins was done in an aqueous medium at pH 8.3 to produce immunogenic and enzymatic conjugates of Amp and Amox. The β-lactam cycle of Amp was shown to remain intact after chemical modification and synthesis of linker conjugates. An immunogenic Amp–thyroglobulin conjugate containing an aromatic linker was used for long-term immunization of rabbits, and polyclonal antibodies thus obtained were found to bind Amp, Amox, and penicillin G with extremely high sensitivity. Amp and Amox conjugates with horseradish peroxidase (HRP) were synthesized and characterized in a competitive protein-binding (receptor) assay and a direct competitive enzyme-linked immunosorbent assay (ELISA). Of the model immunoassay systems tested, the best characteristics were observed for heterologous direct ELISA with polyclonal antibodies and the Amp–HRP conjugate that contained an adipic acid fragment as a linker: the Amp sensitivity was 0.03 ng/mL and IC50 = 0.20 ng/mL.

Direct Conjugation of Penicillins and Cephalosporins with Proteins for Receptor Assays of Beta-Lactam Antibiotics

—Fifteen protein conjugates of penicillins and cephalosporins containing amino- and/or carboxylic groups in the initial structures have been synthesized in the reactions with human serum albumin or ovalbumin using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) or a combination of EDC and N-hydroxysulfosuccinimide at various ratios of the base reagents. A comparative study of conjugates composition and properties has been carried out by UV spectroscopy, mass spectrometry and a ligand-receptor assay. It was shown that the antibiotic residue content of the macromolecules obtained varied from 1 to 22, the beta-lactam cycle remained intact assuring specific interactions of the conjugates with a penicillin-binding protein. In two developed models of receptor bioanalytic systems, an ampicillin conjugate onto a solid phase binds to penicillin-binding protein complexed with a monoclonal antibody, which was detected by an immunoenzyme label in microplate wells or gold nanoparticles on test strips. Conjugated ampicillin binding to the receptor was competitively inhibited by beta-lactam antibiotics added to the liquid phase, and analytical sensitivities relative to penicillin G were 0.05 and 1 ng/mL for microplate and receptor chromatographic systems, respectively.