Maximum Specific Binding Research Articles

Preclinical studies of a PARP targeted, Meitner-Auger emitting, theranostic radiopharmaceutical for metastatic ovarian cancer

Advanced ovarian cancer currently has few therapeutic options. Poly(ADP-ribose) polymerase (PARP) inhibitors bind to nuclear PARP and trap the protein-inhibitor complex to DNA. This work investigates a theranostic PARP inhibitor for targeted radiopharmaceutical therapy of ovarian cancer in vitro and PET imaging of healthy mice in vivo. Methods[77Br]RD1 was synthesized and assessed for pharmacokinetics and cytotoxicity in human and murine ovarian cancer cell lines. [76Br]RD1 biodistribution and organ uptake in healthy mice were quantified through longitudinal PET/CT imaging and ex vivo radioactivity measurements. Organ-level dosimetry following [76/77Br]RD1 administration was calculated using RAPID, an in-house platform for absorbed dose in mice, and OLINDA for equivalent and effective dose in human. ResultsThe maximum specific binding (Bmax), equilibrium dissociation constant (Kd), and nonspecific binding slope (NS) were calculated for each cell line. These values were used to calculate the cell specific activity uptake for cell viability studies. The half maximal effective concentration (EC50) was measured as 0.17 (95 % CI: 0.13–0.24) nM and 0.46 (0.13–0.24) nM for PARP(+) and PARP(−) expressing cell lines, respectively. The EC50 was 0.27 (0.21–0.36) nM and 0.30 (0.22–0.41) nM for BRCA1(−) and BRCA1(+) expressing cell lines, respectively. When measuring the EC50 as a function of cellular activity uptake and nuclear dose, the EC50 ranges from 0.020 to 0.039 Bq/cell and 3.3–9.2 Gy, respectively. Excretion through the hepatobiliary and renal pathways were observed in mice, with liver uptake of 2.3 ± 0.4 %ID/g after 48 h, contributing to estimated absorbed dose values in mice of 19.3 ± 0.3 mGy/MBq and 290 ± 10 mGy/MBq for [77Br]RD1 and [76Br]RD1, respectively. Conclusion[77Br]RD1 cytotoxicity was dependent on PARP expression and independent of BRCA1 status. The in vitro results suggest that [77Br]RD1 cytotoxicity is driven by the targeted Meitner-Auger electron (MAe) radiotherapeutic effect of the agent. Further studies investigating the theranostic potential, organ dose, and tumor uptake of [76/77Br]RD1 are warranted.

Evaluation of Tau Radiotracers in Chronic Traumatic Encephalopathy.

Chronic traumatic encephalopathy (CTE) is a neurologic disorder associated with head injuries, diagnosed by the perivascular accumulation of hyperphosphorylated tau protein (phospho-tau) identified at autopsy. Tau PET radiopharmaceuticals developed for imaging Alzheimer disease are under evaluation for brain injuries. The goal of this study was to conduct a head-to-head invitro evaluation of 5 tau PET radiotracers in subjects pathologically diagnosed with CTE. Methods: Autoradiography was used to assess the specific binding and distribution of 3H-flortaucipir (also known as Tauvid, AV-1451, and T807), 3H-MK-6240 (also known as florquinitau), 3H-PI-2620, 3H-APN-1607 (also known as PM-PBB3 and florzolotau), and 3H-CBD-2115 (also known as 3H-OXD-2115) in fresh-frozen human postmortem CTE brain tissue (stages I-IV). Immunohistochemistry was performed for phospho-tau with AT8, 3R tau with RD3, 4R tau with RD4 and amyloid-β with 6F/3D antibodies. Tau target density (maximum specific binding) was quantified by saturation analysis with 3H-flortaucipir in tissue sections. Results: 3H-flortaucipir demonstrated a positive signal in all CTE cases examined, with varying degrees of specific binding (68.7% ± 10.5%; n = 12) defined by homologous blockade and to a lesser extent by heterologous blockade with MK-6240 (27.3% ± 13.6%; n = 12). The 3H-flortaucipir signal was also displaced by the monoamine oxidase (MAO)-A inhibitor clorgyline (43.9% ± 4.6%; n = 3), indicating off-target binding to MAO-A. 3H-APN-1607 was moderately displaced in homologous blocking studies and was not displaced by 3H-flortaucipir; however, substantial displacement was observed when blocking with the β-amyloid-targeting compound NAV-4694. 3H-MK-6240 and 3H-PI-2620 had negligible binding in all but 2 CTE IV cases, and binding may be attributed to pathology severity or mixed Alzheimer disease/CTE pathology. 3H-CBD-2115 showed moderate binding, displaced under homologous blockade, and aligned with 4R-tau immunostaining. Conclusion: In human CTE tissues, 3H-flortaucipir and 3H-APN-1607 revealed off-target binding to MAO-A and amyloid-β, respectively, and should be considered if these radiotracers are used in PET imaging studies of patients with brain injuries. 3H-MK-6240 and 3H-PI-2620 bind to CTE tau in severe- or mixed-pathology cases, and their respective 18F PET radiotracers warrant further evaluation in patients with severe suspected CTE.

OBTAINING NEUTRON MATTER AND HYPERHEAVY NUCLEI: POSSIBLE QUANTUM-TECHNOLOGICAL INSTRUMENTAL APPROACHES

Possible mechanisms of creation of both hyperheavy nuclei by electron-nuclear collapse and neutron matter by condensation of ultracold neutrons are discussed. The fundamental possibility of the existence of such objects was previously substantiated by A.B.Migdal, who suggested that the known set of proton-neutron nuclei with mass numbers from 0 to 300 and a maximum specific binding energy of about 8 MeV / nucleon at A≈60 corresponds to the first region, beyond which (starting from about the charge Z≈ ( hc/e2 )3/2 ≈1600 ) there is an additional region describing a possible state of nuclear matter, stabilized by a pion condensate. In this region, the maximum specific energy corresponds to ≈15 MeV / nucleon at A ≈ 100000. It is shown that neutron matter can be obtained under certain conditions, and its systematization can be realized as an addition to the Periodic Table. When solving such problems, it becomes quite real to study not only physical, but also chemical, and possibly engineering and technical properties. Analysis shows that the stability of neutron matter at the microlevel is ensured by the Tamm interaction and the Hund beta equilibrium. Such matter can be quite stable not only on the mega-level (neutron stars) due to gravitational interaction, as was a priori assumed earlier, but also on the scale of "ordinary" matter. The process of neutronization is possible not only with critical gravitational interaction, but also by other mechanisms (supercritical increase in the atomic number of elements due to electron-nuclear collapse and condensation of ultracold neutrons), which opens the way to the fundamental possibility of obtaining both neutron matter in laboratory conditions and superheavy nuclei. Based on the works of Migdal, Tamm and Hund, the possibility of the existence of stable neutron matter (with Z >> 175, N >> Z, A> 10 3 -10 5 and a size of 200-300 femtometers and more) is argued at the microlevel, and not only at the mega-level, as is now considered in astrophysics. A critical analysis of the well-established concept of the minimum possible mass of neutron stars is carried out. The following quantum technological approaches to the realization of UCN condensation are proposed: 1. Slow isothermal compression; 2. Refrigerator for dissolving helium-3 and helium-4; 3. Use of a conical concentrator for UCN focusing (Vysotskii cone); 4. Magnetic trap; 5. Additional UCN laser cooling. Neutron matter is considered as a potential cosmological candidate for dark matter. One should take into account the possibility of the formation of fragments of neutron matter as dark matter (neutral, femto-, pico- and nanoscale, the cooling of relics makes it difficult to detect them by now) already at the initial origin of the Universe, which is the dominant process. The observable part of the Universe is formed by the residual part of protons, and then by decayed single neutrons and unstable fragments of neutron matter (with Z> 175, N >> Z, but A <10 3 -10 5 ).

Differential expression and signaling of the human histamine H3 receptor isoforms of 445 and 365 amino acids expressed in human neuroblastoma SH-SY5Y cells

In stably-transfected human neuroblastoma SH-SY5Y cells, we have compared the effect of activating two isoforms of 445 and 365 amino acids of the human histamine H3 receptor (hH3R445 and hH3R365) on [35S]-GTPγS binding, forskolin-induced cAMP formation, depolarization-induced increase in the intracellular concentration of Ca2+ ions ([Ca2+]i) and depolarization-evoked [3 H]-dopamine release. Maximal specific binding (Bmax) of [3 H]-N-methyl-histamine to cell membranes was 953 ± 204 and 555 ± 140 fmol/mg protein for SH-SY5Y-hH3R445 and SH-SY5Y-hH3R365 cells, respectively, with similar dissociation constants (Kd, 0.86 nM and 0.81 nM). The mRNA of the hH3R365 isoform was 40.9 ± 7.9% of the hH3R445 isoform. No differences in receptor affinity were found for the H3R ligands histamine, immepip, (R)(-)-α-methylhistamine (RAMH), A-331440, clobenpropit and ciproxifan. Both the stimulation of [35S]-GTPγS binding and the inhibition of forskolin-stimulated cAMP accumulation by the agonist RAMH were significantly larger in SH-SY5Y-hH3R445 cells ([35S]-GTPγS binding, 158.1 ± 7.5% versus 136.5 ± 3.6% for SH-SY5Y-hH3R365 cells; cAMP accumulation, −74.0 ± 4.9% versus −43.5 ± 5.3%), with no significant effect on agonist potency. In contrast, there were no differences in the efficacy and potency of RAMH to inhibit [3 H]-dopamine release evoked by 100 mM K+ (−18.9 ± 3.0% and −20.5 ± 3.3%, for SH-SY5Y-hH3R445 and SH-SY5Y-hH3R365 cells), or the inhibition of depolarization-induced increase in [Ca2+]i (S2/S1 ratios: parental cells 0.967 ± 0.069, SH-SY5Y-hH3R445 cells 0.639 ± 0.049, SH-SY5Y-hH3R365 cells 0.737 ± 0.045). These results indicate that in SH-SY5Y cells, hH3R445 and hH3R365 isoforms regulate in a differential manner the signaling pathways triggered by receptor activation.

A monoclonal antibody raised against a thermo-stabilised β1-adrenoceptor interacts with extracellular loop 2 and acts as a negative allosteric modulator of a sub-set of β1-adrenoceptors expressed in stable cell lines

Recent interest has focused on antibodies that can discriminate between different receptor conformations. Here we have characterised the effect of a monoclonal antibody (mAb3), raised against a purified thermo-stabilised turkey β1-adrenoceptor (β1AR-m23 StaR), on β1-ARs expressed in CHO-K1 or HEK 293 cells. Immunohistochemical and radioligand-binding studies demonstrated that mAb3 was able to bind to ECL2 of the tβ1-AR, but not its human homologue. Specific binding of mAb3 to tβ1-AR was inhibited by a peptide based on the turkey, but not the human, ECL2 sequence. Studies with [3H]-CGP 12177 demonstrated that mAb3 prevented the binding of orthosteric ligands to a subset (circa 40%) of turkey β1-receptors expressed in both CHO K1 and HEK 293 cells. MAb3 significantly reduced the maximum specific binding capacity of [3H]-CGP-12177 without influencing its binding affinity. Substitution of ECL2 of tβ1-AR with its human equivalent, or mutation of residues D186S, P187D, Q188E prevented the inhibition of [3H]-CGP 12177 binding by mAb3. MAb3 also elicited a negative allosteric effect on agonist-stimulated cAMP responses. The identity of the subset of turkey β1-adrenoceptors influenced by mAb3 remains to be established but mAb3 should become an important tool to investigate the nature of β1-AR conformational states and oligomeric complexes.

REGIONAL DIFFERENCES IN THE TRANSIENT EQUILIBRIUM OF [18F]AV-1451 AND THEIR IMPACT ON TISSUE UPTAKE RATIOS

Absolute quantification of PET ligand uptake requires true equilibrium between tissue and plasma radioactivity. While this can only be achieved using continuous ligand infusion, a ”transient equilibrium” (TrEq) at the maximum specific binding can be identified using a bolus infusion protocol. Theoretically, the TrEq could be used to estimate the specific binding of the tracer, described as the tissue ratio of specific and non-displaceable binding. Deviation from TrEq when estimating tissue ratios, causes over- or underestimation of the binding, and the bias can vary across brain regions and patient groups due to variability in the time point at which TrEq (tTrEq) occurs. We investigated the tTrEq of [18F]AV-1451 across different regions and their impact on simplified quantification approaches. Six patients with probable Alzheimer's disease (AD) (64.8 ± 14.7y) and four cognitively healthy elderly (75.0 ± 3.8y) underwent structural MRI and [18F]AV-1451 dynamic PET scanning over 0–60, 80–140 and 160–180 min post injection together with arterial blood sampling. Tissue radioactivity (CT) curves were generated using the Harvard-Oxford atlas, with non-displaceable binding (CND) represented by the cerebellar cortex. Radioactivity concentration of specifically bound ligand was estimated as CS=CT-CND, with tTrEq determined as dCS/dt=0. Arterial input Logan binding potentials (BPND=VT/VND-1) were used as reference for comparison of different time windows in the estimation of specific binding ratios (SBr=∫ CS dt/∫ CNDdt, i.e. SUVR-1). A range of time windows was examined, including the commonly used interval of 80–100 min post injection. The average tTrEqdiffered between diagnostic groups and brain regions, ranging from 32.5 in the basal ganglia to 137.5 min post injection in occipital and lateral parietal cortex. Later time windows resulted in increasingly overestimated binding in AD patients using SBR, of different magnitude across regions. The tTrEq varies across regions, affecting the agreement between BPND and SBR differentially in different brain regions. Though Logan BPND may itself introduce a negative bias, our findings suggest that there is a regional component to the accuracy of late scan SBR, and they should thus be interpreted with caution when compared across brain regions.

Abstract 2071: SW43-DOX - a potential new targeted drug for the treatment of liver malignancies - An in vitro evaluation of receptor affinity and tumor internalization

Abstract Purpose: The σ2-receptor is known to be overexpressed on many rapidly growing cancer cells, thus represents a tumor biomarker and is potentially attractive for targeted tumor therapy. We have synthesized the conjugate SW43-DOX consisting of the σ2-receptor agonist SW43 and the anticancer agent Doxorubicin (DOX). The purpose of this study was to compare the half maximal effective concentration (EC50) of SW43-DOX and DOX and to evaluate the receptor affinity and internalization of SW43-DOX in hepatocellular carcinoma and other cancer cell lines in vitro. Material & Methods: SW43 was synthesized and conjugated with Doxorubicin (A Chemtek Inc.) For saturation binding & internalization assays, the chelator L-NETA was attached & loaded with Lu177. Hep G2, Hep 3B, Panc-1 & HT-29 cell lines were evaluated. EC50 was assessed by treatment with various concentrations ranging from 0.5-700 μM of SW43-DOX/Doxorubicin (CellTiter-Glo®; Promega). Saturation binding assay: σ2-receptor blocking was achieved with 10-100 μM SW43 for 15 min. at RT prior to adding 1-300 μM SW43-DOX-Lu177 in HBSS buffer (+0.1% BSA) incubating for 3.5h on ice. Internalization assay: After receptor blocking (7.5 μM SW43; 15 min.), cells were incubated with 75 nM SW43-DOX-Lu177 for .5, 1 & 2h at 37°C/5% CO2. The cell surface bound fraction from the internalization assay was scavenged by incubation with HBSS (+20 mM NaOAc; pH4.0; 10 min; 37°C/5% CO2). Cells were lysed with 0.5% SDS, radioactivity measured (Cobra -counter, Packard Bell) & normalized to whole cell protein. Statistics: EC50: Dose response non-linear fitting model; Internalization: ANOVA + Bonferroni post-hoc test, Saturation binding: One site saturation non-linear regression fit model. Prism V6.0. Results: EC50 showed lower needed concentration (μM) for SW43-DOX compared to Doxorubicin: Hep G2 (12.0 (95%CI: 10.3-13.9) vs. 81.5 (95%CI: 66.7-99.5); p<0.001), Hep 3B (8.0 (95%CI: 7.2-9) vs. 17.6 (95%CI: 14.1-22); p<0.001), Panc-1 (21.59 (95%CI: 15.44-30.2) vs. 64.5 (95%CI: 50.3-82.6); p = 0.31), and HT-29 (20.7 (95%CI: 18.3-23.3) vs. 179.8 (95%CI: 65.9-593.5); p<0.001). Maximal specific cell surface binding capacity (Bmax; pmol/mg): Hep G2 (21.5; 95%CI: 14.3-28.6), Hep 3B (35.2; 95%CI: 27.9-42.5), Panc-1 (66.5; 95%CI: 51.6-78.2) & HT-29 (33.4; 95%CI: 8.43-58.5). Specific binding affinity (KD) in nM: Hep G2 (49.2; 95%CI: 2.2-96.3), Hep 3B (40.8; 95%CI: 13.4-68.1), Panc-1 (96.4; 95%CI: 51.4-141.5), HT-29 (31.3; 95%CI: -40.5-84.4). Specific internalization was evident at all time points for all cell lines (p<0.05). Specific internalization (pmol/mg ±SD) after 2h was 29.5 ±6.6 (Panc-1), 10.3 ±1.6 (HT-29), 18.2 ±2.36 (Hep G2) & 30.7 ±8.2 (Hep 3B). Conclusion: SW43-DOX exerts a higher antitumoral effect than Doxorubicin & binds specifically onto the cell surface with high affinity & subsequently specific uptake via the σ2-receptor. Citation Format: Johannes M. Ludwig, Yongkang Gai, Sun Lingyi, Dexing Zeng, Hyun S. Kim. SW43-DOX - a potential new targeted drug for the treatment of liver malignancies - An in vitro evaluation of receptor affinity and tumor internalization. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2071.

Relationship Between Values of Fowlpox ELISA and the Presence of "Takes" After Vaccination.

Values from an ELISA for evaluating the immune response induced by a commercial vaccine against fowlpox virus and the lesion at the site of inoculation (i.e., swelling of the skin or a pox where the vaccine was applied) were compared. The ELISA was carried out with an antigen prepared by precipitation of a cell culture-propagated virus suspension with ammonium sulfate and concentration by centrifugation. A 0.1 M acetate buffer (pH 5) was used as the sensitizing solution for maximum specific binding of the antigen to the microplate plastic well. Four experiments were conducted where the birds were bled once a week before and after vaccination and then were examined simultaneously for evidence of "takes." This study showed that there is a relationship between the ELISA values to the fowlpox vaccine that are considered positive and the presence of postvaccination lesions.

Molecularly imprinted polymer strategies for removal of a genotoxic impurity, 4-dimethylaminopyridine, from an active pharmaceutical ingredient post-reaction stream

Abstract Molecularly imprinted polymers (MIPs) are prepared and evaluated for the effective removal of the genotoxic impurity (GTI), 4-dimethylaminopiridine (DMAP), from a Mometasone furoate (Meta) solution, used as a case study relevant for the pharmaceutical industry. The MIPs formation by bulk polymerization is assessed considering different temperature regimes as well as stoichiometry of template, functional monomer, cross-linker, respectively DMAP, methacrylic acid and ethylene glycol dimethacrylate. A design of experiment (DoE) is performed to establish conditions for a maximum GTI specific binding percentage, validated experimentally at a value of 98% for 5.03 mgGTI/gMIP, for a 256 ppm GTI solution. The MIP robustness and recyclability are successfully evaluated over 6 cycles. Multistep approaches, using MIP alone or in combination with organic solvent nanofiltration (OSN), are discussed aiming to minimize API losses with removal of GTI to reach the threshold of toxicological concern (TTC) for two case studies.

Towards the design of hypolipidaemic peptides: Deoxycholate binding affinity of hydrophobic peptide aggregates of casein plastein

Plastein is a thixotropic product of protease-induced peptide aggregation. This study evaluated the effects of the modified peptide structure of plastein on its affinity for a secondary bile acid. Casein plastein formation resulted in decreased free amino nitrogen and increased mean particle size, surface hydrophobicity and hydrophobic-to-hydrophilic amino acid ratio of the peptides. The net negative surface charge of casein hydrolysate (CH) was decreased (P < 0.05) in crude plastein (CPc) and completely lost in the isolated plastein (CPi) resulting in the formation of insoluble aggregates. The surface and structural changes in CPi were associated with 38.5 ± 0.0% binding of physiological level of sodium deoxycholate, higher (P < 0.05) than the activities of CPc (23.1 ± 0.0%) and CH (12.8 ± 4.4%). Maximum specific ligand binding (Bmax) was higher (P < 0.05) for CPc (0.391 ± 0.021 mM/mg protein) than CH (0.246 ± 0.043 mM/mg protein) and CPi (0.234 ± 0.005 mM/mg protein). However, CPi had the strongest affinity for sodium deoxycholate with the lowest dissociation constant (Kd) of 2.719 ± 0.146 mM, which was 2.5- and 3-folds lower (P < 0.05) than those of CH and CPc, respectively. Understanding deoxycholate-binding affinity of peptides can facilitate the design of potent food-based BA sequestrants for managing hypercholesterolaemia (dyslipidaemia) and associated health conditions.

Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE

EmrE is a member of the small multidrug resistance (SMR) protein family in Escherichia coli. EmrE confers resistance to a wide variety of quaternary cation compounds (QCCs) as an efflux transporter driven by proton motive force. The purification yield of most membrane proteins are challenging because of difficulties in over expressing, isolating and solubilizing them and the addition of an affinity tag often improves purification. The purpose of this study is to compare the structure and function of hexahistidinyl (His6) tagged (T-EmrE) and untagged (UT-EmrE) versions of EmrE. In vivo QCC resistance assays determined that T-EmrE demonstrated reduced resistance as compared to UT-EmrE. We isolated EmrE using the two different purification methods, an organic solvent extraction method used to isolate UT-EmrE and nickel affinity chromatography of T-EmrE. All proteins were solubilized in the same buffered n-dodecyl-β-d-maltopyranoside (DDM) detergent and their conformations were examined in the presence/absence of different QCCs. In vitro analysis of protein multimerization using SDS-Tricine PAGE and dynamic light scattering analysis revealed that both proteins predominated as monomers, but the formation of dimers was more constant and uniform in T-EmrE compared to UT-EmrE. The aromatic residue conformations of both proteins indicate that T-EmrE form is more aqueous exposed than UT-EmrE, but UT-EmrE appeared to have a more dynamic environment surrounding its aromatic residues. Using fluorescence to obtain QCC ligand-binding curves indicated that the two forms had differences in dissociation constants (Kd) and maximum specific one-site binding (Bmax) values for particular QCCs. In vitro analyses of both proteins demonstrated subtle but significant differences in multimerization and QCC binding. In vivo analysis indicates differences caused by the addition of the tag, we also observed differences in vitro that could be a result of the tag and/or the different purification methods.

Novel characteristics of a carbohydrate-binding module 20 from hyperthermophilic bacterium.

In this study, a gene fragment coding carbohydrate-binding module 20 (CBM20) in the amylopullulanase (APU) gene was cloned from the hyperthermophilic bacteria Thermoanaerobacter pseudoethanolicus 39E and expressed in Escherichia coli. The protein, hereafter Tp39E, possesses very low sequence similarity with the CBM20s previously reported and has no starch binding site 2. Tp39E did not demonstrate thermal denaturation at 50 °C; however, thermal unfolding of the protein was observed at 59.5 °C. A binding assay with Tp39E was conducted using various soluble and insoluble substrates, and starch was the best binding polysaccharide. Intriguingly, Tp39E bound, to a lesser extent, to soluble and insoluble xylan as well. The dissociation constant (K d) and the maximum specific binding (B max) of Tp39E to corn starch granules were 0.537 μM and 5.79 μM/g, respectively, at pH 5.5 and 20 °C. 99APU1357 with a Tp39E domain exhibited 2.2-fold greater activity than a CBM20-truncation mutant when starch granules were the substrate. Tp39E was an independently thermostable CBM and had a considerable effect on APU activity in the hydrolysis of insoluble substrates.

English

The aim of the study was to illustrate the radio-receptor assay of beetal recombinant caprine growth hormone (rcGH). Tracer ( 125 I-rcGH) was prepared by iodinating beetal rcGH with iodine-125 and its biological activity was analyzed by rabbit anti-rcGH antibodies. Liver microsomal membranes of the Bovidae species (caprine, ovine and bovine) were prepared to study the binding properties of beetal rcGH. The tracer binding was dependent on receptor protein concentration, tracer counts, temperature, time of incubation and assay pH. The maximum percentage specific binding of the tracer to the microsomal membrane was 45% in 32 h at 4°C. In cross-reactive study, human GH competed and displaced the 125 I-rcGH by binding effectively to the cGH receptor. Scatchard analysis of the rcGH binding suggested a single class of binding site to the caprine microsomal membrane with an affinity of 337.1 ± 82.94 × 10 9 M -1 and capacity of 57.61 ± 4.23 fmol mg -1 while rcGH binding affinity and capacity to the ovine and bovine receptor protein showed 365.4 ± 66.82 × 10 9 M -1 , 57.69 ± 3.23 (fmol mg -1 ) and 392.6 ± 56.25 × 10 9 M -1 , 56.8 ± 2.56 (fmol mg -1 ) respectively. This study provides data for beetal rcGH interaction with microsomal membrane that shall be beneficial to study hormone receptor interactions of other Bovidae species. Keywords: Beetal recombinant caprine growth hormone, Bovidae, iodine-125, microsomal membrane, receptor protein African Journal of Biotechnology , Vol 13(30) 3081-3091

Trichuris suis and Oesophagostomum dentatum Show Different Sensitivity and Accumulation of Fenbendazole, Albendazole and Levamisole In Vitro

BackgroundThe single-dose benzimidazoles used against Trichuris trichiura infections in humans are not satisfactory. Likewise, the benzimidazole, fenbendazole, has varied efficacy against Trichuris suis whereas Oesophagostomum dentatum is highly sensitive to the drug. The reasons for low treatment efficacy of Trichuris spp. infections are not known.MethodologyWe studied the effect of fenbendazole, albendazole and levamisole on the motility of T. suis and O. dentatum and measured concentrations of the parent drug compounds and metabolites of the benzimidazoles within worms in vitro. The motility and concentrations of drug compounds within worms were compared between species and the maximum specific binding capacity (Bmax) of T. suis and O. dentatum towards the benzimidazoles was estimated. Comparisons of drug uptake in living and killed worms were made for both species.Principal findingsThe motility of T. suis was generally less decreased than the motility of O. dentatum when incubated in benzimidazoles, but was more decreased when incubated in levamisole. The Bmax were significantly lower for T. suis (106.6, and 612.7 pmol/mg dry worm tissue) than O. dentatum (395.2, 958.1 pmol/mg dry worm tissue) when incubated for 72 hours in fenbendazole and albendazole respectively. The total drug concentrations (pmol/mg dry worm tissue) were significantly lower within T. suis than O. dentatum whether killed or alive when incubated in all tested drugs (except in living worms exposed to fenbendazole). Relatively high proportions of the anthelmintic inactive metabolite fenbendazole sulphone was measured within T. suis (6–17.2%) as compared to O. dentatum (0.8–0.9%).Conclusion/SignificanceThe general lower sensitivity of T. suis towards BZs in vitro seems to be related to a lower drug uptake. Furthermore, the relatively high occurrence of fenbendazole sulphone suggests a higher detoxifying capacity of T. suis as compared to O. dentatum.

Insulin action in adipose tissue in type 1 diabetes

Background:Insulin action has been reported to be normal in type 1 diabetic patients. However, some studies have reported an insulin resistance state in these patients. The aim of this study was to investigate insulin resistance in a group of type 1 diabetic patients. We studied the insulin action in adipose tissue and analyzed the effects of duration of disease, body mass index (BMI), and glycosylated hemoglobin on insulin action at the receptor and postreceptor levels in adipocytes.Methods:Nine female type 1 diabetic patients with different durations of disease and eight nondiabetic female patients of comparable age and BMI were studied. 125I-insulin binding and U-[14C]-D-glucose transport was measured in a sample of subcutaneous gluteus adipose tissue obtained by open surgical biopsy from each subject.Results:The duration of disease was negatively correlated with both 125I-insulin binding capacity (r = −0.70, P < 0.05) and basal and maximum insulin-stimulated glucose transport (r = −0.87, P < 0.01, and r = −0.88, P < 0.01, respectively). Maximum specific 125I-insulin binding to the receptors in adipocytes was higher in the group of patients with a shorter duration of disease (P < 0.01). Basal and maximum insulin-stimulated glucose transport was significantly higher in the group with less than 5 years of disease (P < 0.01). No correlation was found between BMI and insulin action.Conclusion:Female type 1 diabetic patients have normal insulin action. There is a high glucose uptake in the early phase of the disease, although a longer duration of disease appears to be a contributing factor to a decrease in insulin action in these patients, and involving both receptor and postreceptor mechanisms.

Spin-coated methacrylic acid copolymer thin films for covalent immobilization of small molecules on surface plasmon resonance substrates

Hydrophilic copolymers of methacrylic acid and ethylene glycol dimethacrylate are simply and rapidly prepared as thin films by spin-coating on gold-coated glass slides with a concurrent photo-crosslinking step. Coating techniques were optimized for use on gold surfaces both separately and as part of surface plasmon resonance (SPR) sensor chips. The population of carboxylic acid functional groups as binding sites in the polymer matrix, as reflected in the corresponding hydrophilicity, could be easily adjusted through changes to the stoichiometric ratio of the monomers, allowing for good control of immobilization capacity. The polymers used adhered to the gold surfaces both with and without use of thiol moieties. Coating thickness was measured by ellipsometry and coatings of 30–40 nm thickness were routinely achieved on gold-coated slides. This dimension is dependent on the spin speed and the viscosity of the polymerization mixture applied. The polymers were further characterized by contact angle measurements and infrared spectroscopy before being applied to immobilization of the steroid cortisol in a BIAcore SPR instrument, where binding to a monoclonal antibody was studied and the surface coatings optimized for maximum specific binding capacity. Optimized surfaces could be regenerated and re-used, and have potential applications as immobilization matrices in plasmonic biosensors with a very rapid coating technique.

Removing TRPV1-expressing primary afferent neurons potentiates the spinal analgesic effect of δ-opioid agonists on mechano-nociception

Most δ-opioid receptors are located on the presynaptic terminals of primary afferent neurons in the spinal cord. However, their presence in different phenotypes of primary afferent neurons and their contribution to the analgesic effect of δ-opioid agonists are not fully known. Resiniferatoxin (RTX) is an ultra-potent transient receptor potential vanilloid type 1 channel (TRPV1) agonist and can selectively remove TRPV1-expressing primary afferent neurons. In this study, we determined the role of δ-opioid receptors expressed on TRPV1 sensory neurons in the antinociceptive effect of the δ-opioid receptor agonists [ d-Pen 2, d-Pen 5]-enkephalin and [ d-Ala 2,Glu 4]-deltorphin. Nociception was measured by testing the mechanical withdrawal threshold in the hindpaw of rats. Changes in the δ-opioid receptors were assessed using immunocytochemistry and the [ 3H]-naltrindole radioligand binding. In RTX-treated rats, the δ-opioid receptor on TRPV1-immunoreactive dorsal root ganglion neurons and afferent terminals in the spinal cord was diminished. RTX treatment also significantly reduced the maximal specific binding sites (31%) of the δ-opioid receptors in the dorsal spinal cord. Interestingly, intrathecal injection of [ d-Pen 2, d-Pen 5]-enkephalin or [ d-Ala 2,Glu 4]-deltorphin produced a large and prolonged increase in the nociceptive threshold in RTX-treated rats. These findings indicate that loss of TRPV1-expressing afferent neurons leads to a substantial reduction in presynaptic δ-opioid receptors in the spinal dorsal horn. However, the effect of δ-opioid agonists on mechano-nociception is paradoxically potentiated in the absence of TRPV1-expressing sensory neurons. This information is important to our understanding of the cellular sites and mechanisms underlying the spinal analgesic effect of δ-opioid agonists.

G-Protein inwardly rectifying potassium channels are involved in the hypotensive effect of I1-imidazoline receptor selective ligands

The present study examined the role of G-protein inwardly rectifying potassium (GIRK) channels in the depressor responses elicited by intracisternal injections of imidazoline-like drugs in anesthetized rabbits. Intracisternal injections of the I1-imidazoline receptor (I1R) selective ligands LNP509 (30 microg/kg) and LNP640 (2 microg/kg) (subthreshold doses), and of the GIRK channel opener flupirtine (30 microg/kg) did not affect mean arterial blood pressure (MAP). LNP509 and LNP640, however, elicited substantial depressor responses in rabbits pretreated with flupirtine (-17 +/- 2 and -18 +/- 1 mmHg, respectively, P < 0.05). Injection of higher doses of LNP509 (200 microg/kg) or LNP640 (10 microg/kg) elicited substantial reductions in MAP (-45 +/- 3 and -39 +/- 2 mmHg, respectively, P < 0.05) in naive rabbits. The depressor responses elicited by the higher doses of LNP509 or LNP640 were markedly diminished by pretreatment with the GIRK channel blocker tertiapin-Q (10 microg/kg) (-23 +/- 3 and -26 +/- 2 mmHg, respectively, P < 0.05 compared with nonpretreated rabbits), whereas tertiapin-Q (10 microg/kg) did not affect MAP by itself. Maximal-specific binding (Bmax) of the I1R ligand [I]LNP911 to PC12 cell membranes (296 +/- 59 fmol/mg protein) was enhanced by flupirtine pretreatment whereas it was reduced by tertiapin-Q pretreatment (687 +/- 122 and 68 +/- 21 fmol/mg protein, respectively, P < 0.05 vs. control binding). These findings demonstrate that the modulation of GIRK channels affects I1R's function and raise the possibility that GIRK channels, and I1Rs are parts of a single proteic complex.

Charge modification at conserved positively charged residues of fatty acid binding protein (FABP) from the giant liver fluke Fasciola gigantica: its effect on oligomerization and binding properties

Fatty acid binding proteins (FABPs) are capable of binding hydrophobic ligands with high affinity; thereby facilitating the cellular uptake and intracellular trafficking of fatty acids. In this study, functional characteristics of a cytoplasmic FABP from the giant liver fluke Fasciola gigantica (FgFABP) were determined. Binding of a fluorescent fatty acid analogue 11-[[5-dimethy aminonaphtalene-1-sulphonyl] amino] undecanoic acid (DAUDA) to FgFABP resulted in changes in the emission spectrum. The optimal excitation wavelength and maximum emission of fluorescence for binding activities with DAUDA were 350 nm and 550 nm, respectively. The binding activity for DAUDA was determined from titration experiments and revealed a Kd value of 2.95+/-0.54 microM. Furthermore, we found that cross-linking profile of FgFABP with dithiobis-(succinimidylpropionate) (DSP) in the presence of DAUDA resulted in increased formation of higher-ordered oligomers compared to that in the absence of DAUDA. We also replaced five highly conserved positively charged residues (K9, K58, K91, R107 and K131) with alanine and studied their oligomerization and binding properties of the modified FgFABPs. The obtained data demonstrate that these residues do not appear to be involved in oligomerization. However, the K58A and R107A substitutions exhibited a reduction in binding affinities. K91A and R107A revealed an increase in maximal specific binding.

Glucocorticoid Resistance following Herpes Simplex-1 Infection: Role of Hippocampal Glucocorticoid Receptors

Herpes simplex-1 (HSV-1) is a sporadic cause of viral encephalitis. We have previously demonstrated that corneal HSV inoculation markedly activates the hypothalamo-pituitary-adrenal (HPA) axis. This activation depends on host derived brain interleukine-1 and was resistant to pretreatment with dexamethasone (dex), possibly because immune factors such as pro-inflammatory cytokines can modify the binding capacity of glucocorticoids in the hippocampus. In the present study, we examined whether resistance of the HPA axis activation following intracerebral HSV-1 infection to dex-induced suppression is associated with modifications in hippocampal or pituitary glucocorticoids (GC) receptors or GC receptors in cultured astrocytes. Male rats were injected intracerebroventricularly with purified HSV-1 or vehicle. 48 h later, dex or vehicle was injected intraperitoneally. Rats were sacrificed 3.5 h later. ACTH and corticosterone (CS) were measured in the serum. Specific binding of ³H-dex was measured in the cytosolic fraction of the hippocampus and the pituitary. Dex failed to reduce ACTH and CS responses to HSV-1 infection. In contrast, dex significantly reduced ACTH and CS responses to acoustic neural stimuli. Infection with HSV-1 markedly reduced the hippocampal maximal specific binding of dex with no effect on the dissociation constant (K_d) values. HSV-1 had no effect on the binding of dex in the pituitary. Infection of cultured astrocytes with HSV-1 also reduced the maximal specific binding of dex, but increased the K_dvalue. The results suggest that HSV-1 induced GC resistance may be mediated by downregulation of GC receptors in hippocampal tissue. These results may clarify a mechanism responsible for GC resistance following immune challenges.