Corrigendum to "Large Gliadin Peptides Detected in the Pancreas of NOD and Healthy Mice following Oral Administration".

[This corrects the article DOI: 10.1155/2016/2424306.].

Large Gliadin Peptides Detected in the Pancreas of NOD and Healthy Mice following Oral Administration.

Gluten promotes type 1 diabetes in nonobese diabetic (NOD) mice and likely also in humans. In NOD mice and in non-diabetes-prone mice, it induces inflammation in the pancreatic lymph nodes, suggesting that gluten can initiate inflammation locally. Further, gliadin fragments stimulate insulin secretion from beta cells directly. We hypothesized that gluten fragments may cross the intestinal barrier to be distributed to organs other than the gut. If present in pancreas, gliadin could interact directly with the immune system and the beta cells to initiate diabetes development. We orally and intravenously administered 33-mer and 19-mer gliadin peptide to NOD, BALB/c, and C57BL/6 mice and found that the peptides readily crossed the intestinal barrier in all strains. Several degradation products were found in the pancreas by mass spectroscopy. Notably, the exocrine pancreas incorporated large amounts of radioactive label shortly after administration of the peptides. The study demonstrates that, even in normal animals, large gliadin fragments can reach the pancreas. If applicable to humans, the increased gut permeability in prediabetes and type 1 diabetes patients could expose beta cells directly to gliadin fragments. Here they could initiate inflammation and induce beta cell stress and thus contribute to the development of type 1 diabetes.

The labeling of unsaturated γ-hydroxybutyric acid by heavy isotopes of hydrogen: iridium complex-mediated H/D exchange by C─H bond activation vs reduction by boro-deuterides/tritides.

3-Hydroxycyclopent-1-ene-1-carboxylic acid (HOCPCA (1)) is a potent ligand for high-affinity γ-hydroxybutyric acid binding sites in the central nervous system. Various approaches to the introduction of a hydrogen label onto the HOCPCA skeleton are reported. The outcomes of the feasible C─H activation of olefin carbon (C-2) by iridium catalyst are compared with the reduction of the carbonyl group (C-3) by freshly prepared borodeuterides. The most efficient iridium catalysts proved to be Kerr bulky phosphine N-heterocyclic species providing outstanding deuterium enrichment (up to 91%) in a short period of time. The highest deuterium enrichment (>99%) was achieved through the reduction of ketone precursor 2 by lithium trimethoxyborodeuteride. Hence, analogical conditions were used for the tritiation experiment. [3 H]-HOCPCA selectively labeled on the position C-3 was synthetized with radiochemical purity >99%, an isolated yield of 637 mCi and specific activity = 28.9 Ci/mmol.

Bioavailability Studies and in†vitro Profiling of the Selective Excitatory Amino Acid Transporter Subtype†1 (EAAT1) Inhibitor UCPH-102.

Although the selective excitatory amino acid transporter subtype 1 (EAAT1) inhibitor UCPH-101 has become a standard pharmacological tool compound for in vitro and ex vivo studies in the EAAT research field, its inability to penetrate the blood-brain barrier makes it unsuitable for in vivo studies. In the present study, per os (p.o.) administration (40 mg kg(-1) ) of the closely related analogue UCPH-102 in rats yielded respective plasma and brain concentrations of 10.5 and 6.67 µm after 1 h. Three analogue series were designed and synthesized to improve the bioavailability profile of UCPH-102, but none displayed substantially improved properties in this respect. In vitro profiling of UCPH-102 (10 µm) at 51 central nervous system targets in radioligand binding assays strongly suggests that the compound is completely selective for EAAT1. Finally, in a rodent locomotor model, p.o. administration of UCPH-102 (20 mg kg(-1) ) did not induce acute effects or any visible changes in behavior.

Radiosynthesis and in vitro validation of (3)H-NS14492 as a novel high affinity alpha7 nicotinic receptor radioligand.

The neuronal α7 nicotinic acetylcholine receptor is a homo-pentameric ligand-gated ion channel that is a promising drug target for cognitive deficits in Alzheimer׳s disease and schizophrenia. We have previously described (11)C-NS14492 as a suitable agonist radioligand for in vivo positron emission tomography (PET) occupancy studies of the α7 nicotinic receptor in the pig brain. In order to investigate the utility of the same compound for in vitro studies, (3)H-NS14492 was synthesized and its binding properties were characterized using in vitro autoradiography and homogenate binding assays in pig frontal cortex. (3)H-NS14492 showed specific binding to α7 nicotinic receptors in autoradiography, revealing a dissociation constant (Kd) of 2.1±0.7nM and a maximum number of binding sites (Bmax) of 15.7±2.0fmol/mg tissue equivalent. Binding distribution was similar to that of another selective ligand (125)I-α-bungarotoxin ((125)I-BTX) in autoradiography, and unlabeled NS14492 displaced (125)I-BTX with an inhibition constant (Ki) of 23nM. (3)H-NS14492 bound to α7 nicotinic receptors in homogenized pig frontal cortex with a Kd of 0.8±0.3nM and a Bmax of 30.2±11.6fmol/mg protein. This binding assay further revealed the Ki rank order for a number of α7 nicotinic receptor agonists, and positive allosteric modulators (PAMs). Further, we saw increased binding of (3)H-NS14492 to pig frontal cortex membranes when co-incubated with PNU-120596, a type II PAM. Taken together, these findings show that (3)H-NS14492 is a useful new in vitro radioligand for the pig α7 nicotinic receptor.

Engineered α4ß2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface.

The nicotinic acetylcholine receptor α4ß2 is important for normal mammalian brain function and is known to express in two different stoichiometries, (α4)2(ß2)3 and (α4)3(ß2)2. While these are similar in many aspects, the (α4)3(ß2)2 stoichiometry differs by harboring a third orthosteric acetylcholine binding site located at the α4-α4 interface. Interestingly, the third binding site has, so far, only been documented using electrophysiological assays, actual binding affinities of nicotinic receptor ligands to this site are not known. The present study was therefore aimed at determining binding affinities of nicotinic ligands to the α4-α4 interface. Given that epibatidine shows large functional potency differences at α4-ß2 vs. α4-α4 interfaces, biphasic binding properties would be expected at (α4)3(ß2)2 receptors. However, standard saturation binding experiments with [(3)H]epibatidine did not reveal biphasic binding under the conditions utilized. Therefore, an engineered ß2 construct (ß2(HQT)), which converts the ß(-) face to resemble that of an α4(-) face, was utilized to create (α4)3(ß2(HQT))2 receptors harboring three α4-α4 interfaces. With this receptor, low affinity binding of epibatidine with a Kd of ∼5 nM was observed in sharp contrast to a Kd value of ∼10 pM observed for wild-type receptors. A strong correlation between binding affinities at the (α4)3(ß2(HQT))2 receptor and functional potencies at the wild-type receptor of a range of nicotinic ligands highlighted the validity of using the mutational approach. Finally, large differences in activities at α4-ß2 vs. α4-α4 interfaces were observed for structurally related agonists underscoring the need for establishing all binding parameters of compounds at α4ß2 receptors.

New synthesis and tritium labeling of a selective ligand for studying high-affinity γ-hydroxybutyrate (GHB) binding sites.

3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [(3)H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity, and we demonstrate in vivo brain penetration. In vitro characterization of [(3)H]-1 binding shows high specificity to the high-affinity GHB binding sites.

Design, synthesis, and pharmacological characterization of N- and O-substituted 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-d]azepin-3-ol analogues: novel 5-HT(2A)/5-HT(2C) receptor agonists with pro-cognitive properties.

The isoxazol-3-one tautomer of the bicyclic isoxazole, 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-d]azepin-3-ol (THAZ), has previously been shown to be a weak GABA(A) and glycine receptor antagonist. In the present study, the potential in this scaffold has been explored through the synthesis and pharmacological characterization of a series of N- and O-substituted THAZ analogues. The analogues N-Bn-THAZ (3d) and O-Bn-THAZ (4d) were found to be potent agonists of the human 5-HT(2A) and 5-HT(2C) receptors. Judging from an elaborate pharmacological profiling at numerous other CNS targets, the 3d analogue appears to be selective for the two receptors. Administration of 3d substantially improved the cognitive performance of mice in a place recognition Y-maze model, an effect fully reversible by coadministration of the selective 5-HT(2C) antagonist SB242084. In conclusion, as novel bioavailable cognitive enhancers that most likely mediate their effects through 5-HT(2A) and/or 5-HT(2C) receptors, the isoxazoles 3d and 4d constitute interesting leads for further medicinal chemistry development.

Novel radioiodinated {gamma}-hydroxybutyric acid analogues for radiolabeling and Photolinking of high-affinity {gamma}-hydroxybutyric acid binding sites.

γ-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low- and high-affinity sites in the mammalian brain. To target the specific GHB binding sites, we have developed a (125)I-labeled GHB analog and characterized its binding in rat brain homogenate and slices. Our data show that [(125)I]4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate ([(125)I]BnOPh-GHB) binds to one site in rat brain cortical membranes with low nanomolar affinity (K(d), 7 nM; B(max), 61 pmol/mg protein). The binding is inhibited by GHB and selected analogs, but not by γ-aminobutyric acid. Autoradiography using horizontal slices from rat brain demonstrates the highest density of binding in hippocampus and cortical regions and the lowest density in the cerebellum. Altogether, the findings correlate with the labeling and brain regional distribution of high-affinity GHB sites or [(3)H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid ([(3)H]NCS-382) binding sites. Using a (125)I-labeled photoaffinity derivative of the new GHB ligand, we have performed denaturing protein electrophoresis and detected one major protein band with an apparent mass of 50 kDa from cortical and hippocampal membranes. [(125)I]BnOPh-GHB is the first reported (125)I-labeled GHB radioligand and is a useful tool for in vitro studies of the specific high-affinity GHB binding sites. The related photoaffinity linker [(125)I]4-hydroxy-4-[4-(2-azido-5-iodobenzyloxy)phenyl]butanoate can be used as a probe for isolation of the elusive GHB binding protein.

Design, synthesis, and in vitro pharmacology of new radiolabeled gamma-hydroxybutyric acid analogues including photolabile analogues with irreversible binding to the high-affinity gamma-hydroxybutyric acid binding sites.

Gamma-hydroxybutyric acid (GHB) is a psychotropic compound endogenous to the brain. Despite its potential physiological significance, the complete molecular mechanisms of action remain unexplained. To facilitate the isolation and identification of the high-affinity GHB binding site, we herein report the design and synthesis of the first (125)I-labeled radioligands in the field, one of which contains a photoaffinity label which enables it to bind irreversibly to the high-affinity GHB binding sites.

Analysis of bacterial RNase P RNA and protein interaction by a magnetic biosensor technique.

A magnetic sensor technique was applied to analyze the interaction of immobilized bacterial RNase P protein and 3'-biotinylated RNase P RNA bound to streptavidin-coated magnetic beads. Our measurements with three types of beads from different suppliers resulted in K(d) values of about 1-2 nM (at 4.5 mM Mg(2+) and 150 mM NH(4)(+)) for Escherichia coli RNase P RNA and protein, consistent with previous analyses using different techniques. We further measured affinity of the E. coli RNase P protein to chimeric RNase P RNA variants, consisting of an E. coli specificity domain and an engineered archaeal catalytic domain. A "bacterial-like" 1-bp insertion and 2-nt deletion in the helix P2/P3 region largely improved affinity, providing independent evidence that these elements are crucial for interaction of the two RNase P subunits. Moreover, our study documents that the properties of the streptavidin-coated magnetic beads decide on success or failure of the technique.

A haem cofactor is required for redox and light signalling by the AppA protein of Rhodobacter sphaeroides.

The AppA protein of Rhodobacter sphaeroides is unique in its ability to sense and transmit redox signals as well as light signals. By functioning as antagonist to the PpsR transcriptional repressor, it regulates the expression of photosynthesis genes in response to these environmental stimuli. Here we show binding of the cofactor haem to a domain in the C-terminal part of AppA and redox activity of bound haem. This is supported by the findings that: (i) the C-terminal domain of AppA (AppADeltaN) binds to haemin agarose, (ii) AppADeltaN isolated from Escherichia coli shows absorbance at 411 nm and absorbances at 424 nm and 556 nm after reduction with dithionite and (iii) AppADeltaN can be reconstituted with haem in vitro. Expression of AppA variants in R. sphaeroides reveals that the haem binding domain is important for normal expression levels of photosynthesis genes and for normal light regulation in the presence of oxygen. The haem cofactor affects the interaction of the C-terminal part of AppA to PpsR but also its interaction to the N-terminal light sensing AppA-BLUF domain. Based on this we present a model for the transmission of light and redox signals by AppA.

Magnetic biosensor for the detection of Yersinia pestis.

A novel type of magnetic-beads based magnetic biosensor is described for the detection of Yersinia pestis. Experiments were performed with the antigen fraction F1 of these bacteria. The magnetic sensor platform offers easy and reliable detection of Y. pestis by the use of magnetic beads for labelling and quantification in a single step due to their paramagnetic features. The system uses antiYPF1 antibodies as capture element on ABICAP columns as core element of the magnetic sensor. Several immobilization methods for antibodies on polyethylene were exploited. The established biosensor has a linear detection range of 25-300 ng/ml Y. pestis antigen F1 and a detection limit of 2.5 ng/ml in buffer and human blood serum. The presented sensor system is small, simple, portable and therefore usable as off-lab detection unit for medical and warfare analytes.

CRP determination based on a novel magnetic biosensor.

The c-reactive protein (CRP) is a very significant human blood marker for inflammatory processes and is routinely determined for many clinical purposes. The widespread and well established detection method for this approximately 115 kDa hepatic protein is the high-sensitivity ELISA assay (hsCRP-ELISA) in blood serum. New approaches in medical CRP diagnosis (e.g. for CVD, inflammatory bowel disease) require rapid quantification in native matrices. A novel CRP determination method based on magnetic detection is described and tested for human blood serum, saliva and urine. The detection principle is based on two different anti-CRP antibodies (monoclonal, IgG) for CRP trapment and labelling. The linear detection range of this immunosensor ranged from 25 ng/ml to 2.5 microg/ml and is therefore much more sensitive than typical hsCRP-ELISA-assays.

SPR-based immunosensor for the CRP detection--a new method to detect a well known protein.

The c-reactive protein (CRP) is one of the significant human blood serum markers for inflammatory processes. The serum presence of this hepatic approximately 115 kDa protein of five identical subunits accompanies several diseases (e.g. CVD, inflammatory bowel diseases) and is nowadays detected by high-sensitivity ELISA assays in blood serum. To enable CRP detection in other matrices, an SPR-based (surface plasmon resonance) immunosensor for the CRP detection has been established. A linear detection range of 2-5 microg CRP per ml was found, using two different antiCRP antibodies (monoclonal, IgG) for CRP trapment and detection. Furthermore, the kinetic antibody association and dissociation constants of one antibody (antiCRP, clone C2) could be determined.

Isopropyl alcohol intoxication in a neonate through chronic dermal exposure: a complication of a culturally-based umbilical care practice.

A 21-day-old boy presented to our emergency department hypotonic, lethargic, and intermittently unresponsive to pain. A workup for ketoacidosis, sepsis, and central nervous system hemorrhage was negative. A urine drug screen collected eight hours after hospitalization showed 39 mg/dl of isopropyl alcohol and 76 mg/dl of acetone. The first serum drug analysis was not performed until 18 hours after admission, at a time when there had been clinical improvement. The isopropyl alcohol concentration was 8 mg/dl, and the acetone concentration was 203 mg/dl. Management was supportive, and the patient stabilized. He was discharged from the hospital in good health in three days. A further review of the history showed no evidence for an oral exposure to isopropyl alcohol. However, since leaving the maternity hospital the mother had been applying gauze pads or cotton balls soaked with isopropyl alcohol to the umbilicus with every diaper change. We conclude that the child suffered from an isopropyl alcohol intoxication that occurred by absorption through the umbilical area.

The beneficial effects of a thromboxane receptor antagonist on spinal cord perfusion following experimental cord injury.

The eicosanoids thromboxane A2 and prostacyclin have opposing actions causing vasoconstriction and vasodilation respectively. The ratio of these two eicosanoids is thus an important determinant of circulatory homeostasis. An increase in this ratio occurs in certain inflammatory conditions with dramatic consequences in organ perfusion. In spinal cord trauma, in addition to direct physical perturbation of the spinal cord, it is likely that further structural and functional loss occurs as a result of decreased tissue perfusion precipitated by an increase in the thromboxane/prostacyclin ratio. This study evaluated hemodynamics and organ perfusion, 3 h following 24 g-cm spinal cord trauma in the rat. The role of thromboxane was investigated with an inhibitor of thromboxane synthesis (Dazoxiben) and with a receptor antagonist (13-APT). Cardiac output and blood pressure were unaffected by Dazoxiben, 13-APT, or spinal cord trauma. Injury effected approximately a 40% decrease in spinal cord perfusion from 0.41 to 0.25 ml/min/g which was not improved by the thromboxane synthase inhibitor, Dazoxiben. 13-ATP completely abrogated the decline in spinal cord blood flow flowing injury. Perfusion of other selected organs demonstrated little change as a result of the spinal trauma. Brain flow remained constant at 0.78 ml/min/g brain. Coronary blood flow, however, declined from 3.2 to 2.0 ml/min/g heart tissue. The data suggest consideration of the importance of thromboxane in therapeutic attempts to reduce secondary injury arising in spinal cord trauma.

A method to measure the degree of control per se in the oral glucose tolerance test.

Using the glucose and insulin values from a 5-hr oral glucose tolerance test, nine quantitative measures have been developed to separate normal, "flat-curve," and non-insulin-dependent diabetic (NIDD) patients. The purpose of these measures is to quantify per se the degree of control operating in glucose homeostasis. A control index which is based upon Swan's minimizing principle and uses only the glucose values was successful in assessing the degree of control operating in glucose homeostasis.

A comparative study of serum ultrafiltrable, ionized, and total calcium in the diagnosis of primary hyperparathyroidism in patients with intermittent or no elevation in total calcium.

Measurement of serum ionized calcium has been shown to be more sensitive a method of diagnosing primary hyperparathyroidism than total calcium in patients with subtle or intermittent elevations of total calcium. The measurement of ionized calcium, however, is technically difficult. The measurement of serum ultrafiltrable calcium would circumvent technical difficulties because atomic absorption spectroscopy would be used to measure the calcium of a filtrate produced by passing serum through a filter which excludes protein-complexed calcium (Worthington ultrafree filter). The normal range for ultrafiltrable calcium (4.7 to 6.8 mg/dl) was determined in 138 patients by nonlinear least-squares analysis and chart review. The serum concentration of ultrafiltrable calcium correlated well with ionized calcium (r = 0.91). Previous studies have demonstrated no benefit in measuring ionized calcium, as opposed to total calcium, in the diagnosis of primary hyperparathyroidism unless there was subtle, intermittent, or no elevation of the total calcium. This comparative study of ultrafiltrable, ionized, and total calcium was, therefore, done in six patients with primary hyperparathyroidism who exhibited intermittent, minimal, or no elevations in serum total calcium. All six patients had symptoms referrable to hyperparathyroidism. All six underwent parathyroid surgery, and a parathyroid adenoma was found in each case. These six patients had a total of 24 concurrent preoperative determinations of ionized, ultrafiltrable, and total calcium levels. The total calcium value was elevated in only 9 of these 24 determinations (38%), ultrafiltrable calcium was elevated in 15 (63%), and ionized calcium was elevated in 23 (96%). The values of ionized calcium were elevated more frequently than both total calcium (p less than 0.0005) and ultrafiltrable calcium (p less than 0.025). The values for ultrafiltrable calcium were more frequently elevated than those for total calcium; this difference, however, was not significant. This study confirms our previous reports showing that ionized calcium is a more sensitive indicator of primary hyperparathyroidism in patients with intermittent or borderline elevation of the total calcium and extends those observations to show that ionized calcium is also a more sensitive indicator of primary hyperparathyroidism than ultrafiltrable calcium in this group of patients.