Low Physiological Concentrations Research Articles

Positive GABAA receptor modulating steroids and their antagonists: Implications for clinical treatments.

GABA is the main inhibitory neurotransmitter in the brain and GABAergic transmission has been shown to be of importance for regulation of mood, memory and food intake. The progesterone metabolite allopregnanolone (Allo) is a positive GABAA receptor modulating steroid with potent effects. In humans, disorders such as premenstrual dysphoric disorder (PMDD), hepatic encephalopathy and polycystic ovarian syndrome are associated with elevated Allo levels and increased negative mood, disturbed memory and increased food intake in some individuals. This is surprising because Allo shares many properties with benzodiazepines and is mainly considered to be anxiolytic and anti-depressant. However, it is well established that, in certain individuals, GABAA receptor activating compounds could have paradoxical effects and thus be anxiogenic in low physiological plasma concentrations but anxiolytic at high levels. We have demonstrated that isoallopregnanolone (Isoallo), the 3β-OH sibling of Allo, functions as a GABAA receptor modulating steroid antagonist (GAMSA) but without any effects of its own on GABAA receptors. The antagonistic effect is noted in most GABAA subtypes investigated in vitro to date. In vivo, Isoallo can inhibit Allo-induced anaesthesia in rats, as well as sedation or saccadic eye velocity in humans. Isoallo treatment has been studied in women with PMDD. In a first phase II study, Isoallo (Sepranolone; Asarina Pharma) injections significantly ameliorated negative mood in women with PMDD compared with placebo. Several GAMSAs for oral administration have also been developed. The GAMSA, UC1011, can inhibit Allo induced memory disturbances in rats and an oral GAMSA, GR3027, has been shown to restore learning and motor coordination in rats with hepatic encephalopathy. In humans, vigilance, cognition and pathological electroencephalogram were improved in patients with hepatic encephalopathy on treatment with GR3027. In conclusion GAMSAs are a new possible treatment for disorders and symptoms caused by hyperactivity in the GABAA system.

Femtomolar Detection of Thrombin in Serum and Cerebrospinal Fluid via Direct Electrocatalysis of Oxygen Reduction by the Covalent G4-Hemin-Aptamer Complex.

Thrombin, a serine protease playing a central role in the coagulation cascade reactions and a potent neurotoxin produced by injured brain endothelial cells, is a recognized cardiac biomarker and a critical biomarker for Alzheimer's disease development. Both in vivo and in vitro, its low physiological concentrations and nonspecific binding of other components of physiological fluids complicate electroanalysis of thrombin. Here, femtomolar levels of thrombin in serum and an artificial cerebrospinal fluid (CSF) were detected by the indicator-free electrochemical methodology exploiting the O2 reduction reaction directly, with no electron transfer mediators, electrocatalyzed by the covalent G4-hemin DNAzyme complex naturally self-assembling upon thrombin binding to the hemin-modified 29-mer DNA aptamer sequence tethered to gold via an alkanethiol linker. Coadsorbed PEG inhibited nonspecific protein binding and allowed the sought signal resolution. The proposed assay exploiting the "oxidase" activity of G4-hemin DNAzyme does not require any coreactants necessary for the traditional peroxidase activity-based assays with this DNAzyme, such as H2O2 and redox mediators, or solution deaeration and allows fast, overall 30 min analysis of thrombin in aerated buffer, CSF, and 1% human serum solutions. This pioneer approach exploiting the oxidase activity G4-hemin DNAzyme is simple, sensitive, and selective and represents a new tool for ultrasensitive electrocatalytic assays based on simple and efficient O2-dependent DNAzyme labels.

Evaluation of the compounds commonly known as superoxide dismutase and catalase mimics in cellular models

Oxidative stress that results from an imbalance between the concentrations of reactive species (RS) and antioxidant defenses is associated with many pathologies. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase are among the key enzymes that maintain the low nanomolar physiological concentrations of superoxide and hydrogen peroxide. The increase in the levels of these species and their progeny could have deleterious effects. In this context, chemists have developed SOD and CAT mimics to supplement them when cells are overwhelmed with oxidative stress. However, the beneficial activity of such molecules in cells depends not only on their intrinsic catalytic activities but also on their stability in biological context, their cell penetration and their cellular localization. We have employed cellular assays to characterize several compounds that possess SOD and CAT activities and have been frequently used in cellular and animal models. We used cellular assays that address SOD and CAT activities of the compounds. Finally, we determined the effect of compounds on the suppression of the inflammation in HT29-MD2 cells challenged by lipopolysaccharide. When the assay requires penetration inside cells, the SOD mimics Mn(III) meso-tetrakis(N-(2′-n-butoxyethyl)pyridinium-2-yl)porphyrin (MnTnBuOE-2-PyP5+) and Mn(II) dichloro[(4aR,13aR,17aR,21aR)-1,2,3,4,4a,5,6,12,13,13a,14,15,16,17,17a,18,19,20,21,21a-eicosahydro-11,7-nitrilo-7Hdibenzo[b,h] [1,4, 7,10] tetraazacycloheptadecine-κN5,κN13,κN18,κN21,κN22] (Imisopasem manganese, M40403, CG4419) were found efficacious at 10 μM, while Mn(II) chloro N-(phenolato)-N,N′-bis[2-(N-methyl-imidazolyl)methyl]-ethane-1,2-diamine (Mn1) requires an incubation at 100 μM. This study thus demonstrates that MnTnBuOE-2-PyP5+, M40403 and Mn1 were efficacious in suppressing inflammatory response in HT29-MD2 cells and such action appears to be related to their ability to enter the cells and modulate reactive oxygen species (ROS) levels.

An ESIPT-based colorimetric and fluorescent probe with large Stokes shift for the sensitive detection of hypochlorous acid and its bioimaging in cells

Hypochlorous acid (HOCl), with a low physiological concentration, plays a vital role in killing the pathogens and anti-inflammation in the human immune system.

The interaction of chondroitin sulfate with a lipid monolayer observed by using nonlinear vibrational spectroscopy.

The first vibrational sum-frequency generation (VSFG) spectra of chondroitin sulfate (CS) interacting with dipalmitoyl phosphatidylcholine (DPPC) at air–liquid interface are reported here, collected at a laser repetition rate of 100 kHz. By studying the VSFG spectra in the regions of 1050–1450 cm−1, 2750–3180 cm−1, and 3200–3825 cm−1, it was concluded that in the presence of Ca2+ ions, the head groups together with the head-group-bound water molecules in the DPPC monolayer are strongly influenced by the interaction with CS, while the organization of the phospholipid tails remains mostly unchanged. The interactions were observed at a CS concentration below 200 nM, which exemplifies the potential of VSFG in studying biomolecular interactions at low physiological concentrations. The VSFG spectra recorded in the O–H stretching region at chiral polarization combination imply that CS molecules are organized into ordered macromolecular superstructures with a chiral secondary structure.

Cholinergic modulation of Up-Down states in the mouse medial entorhinal cortex in vitro.

Cholinergic tone is high during wake and rapid eye movement sleep and lower during slow wave sleep (SWS). Nevertheless, the low tone of acetylcholine during SWS modulates sharp wave ripple incidence in the hippocampus and slow wave activity in the neocortex. Linking the hippocampus and neocortex, the medial entorhinal cortex (mEC) regulates the coupling between these structures during SWS, alternating between silent Down states and active Up states, which outlast neocortical ones. Here, we investigated how low physiological concentrations of acetylcholine (ACh; 100-500nM) modulate Up and Down states in a mEC slice preparation. We find that ACh has a dual effect on mEC activity: it prolongs apparent Up state duration as recorded in individual cells and decreases the total synaptic charge transfer, without affecting the duration of detectable synaptic activity. The overall outcome of ACh application is excitatory and we show that ACh increases Up state incidence via muscarinic receptor activation. The mean firing rate of principal neurons increased in around half of the cells while the other half showed a decrease in firing rate. Using two-photon calcium imaging of population activity, we found that population-wide network events are more frequent and rhythmic during ACh and confirmed that ACh modulates cell participation in these network events, consistent with a role for cholinergic modulation in regulating information flow between the hippocampus and neocortex during SWS.

A dynamic role for dopamine receptors in the control of mammalian spinal networks

Dopamine is well known to regulate movement through the differential control of direct and indirect pathways in the striatum that express D1 and D2 receptors respectively. The spinal cord also expresses all dopamine receptors; however, how the specific receptors regulate spinal network output in mammals is poorly understood. We explore the receptor-specific mechanisms that underlie dopaminergic control of spinal network output of neonatal mice during changes in spinal network excitability. During spontaneous activity, which is a characteristic of developing spinal networks operating in a low excitability state, we found that dopamine is primarily inhibitory. We uncover an excitatory D1-mediated effect of dopamine on motoneurons and network output that also involves co-activation with D2 receptors. Critically, these excitatory actions require higher concentrations of dopamine; however, analysis of dopamine concentrations of neonates indicates that endogenous levels of spinal dopamine are low. Because endogenous levels of spinal dopamine are low, this excitatory dopaminergic pathway is likely physiologically-silent at this stage in development. In contrast, the inhibitory effect of dopamine, at low physiological concentrations is mediated by parallel activation of D2, D3, D4 and α2 receptors which is reproduced when endogenous dopamine levels are increased by blocking dopamine reuptake and metabolism. We provide evidence in support of dedicated spinal network components that are controlled by excitatory D1 and inhibitory D2 receptors that is reminiscent of the classic dopaminergic indirect and direct pathway within the striatum. These results indicate that network state is an important factor that dictates receptor-specific and therefore dose-dependent control of neuromodulators on spinal network output and advances our understanding of how neuromodulators regulate neural networks under dynamically changing excitability.

Lipid kinases PIP5K7 and PIP5K9 are required for polyamine-triggered K+ efflux in Arabidopsis roots.

SUMMARYPolyamines, such as putrescine, spermidine and spermine (Spm), are low‐molecular‐weight polycationic molecules present in all living organisms. Despite their implication in plant cellular processes, little is known about their molecular mode of action. Here, we demonstrate that polyamines trigger a rapid increase in the regulatory membrane lipid phosphatidylinositol 4,5‐bisphosphate (PIP2), and that this increase is required for polyamine effects on K+ efflux in Arabidopsis roots. Using in vivo 32Pi‐labelling of Arabidopsis seedlings, low physiological (μm) concentrations of Spm were found to promote a rapid PIP2 increase in roots that was time‐ and dose‐dependent. Confocal imaging of a genetically encoded PIP2 biosensor revealed that this increase was triggered at the plasma membrane. Differential 32Pi‐labelling suggested that the increase in PIP2 was generated through activation of phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K) activity rather than inhibition of a phospholipase C or PIP2 5‐phosphatase activity. Systematic analysis of transfer DNA insertion mutants identified PIP5K7 and PIP5K9 as the main candidates involved in the Spm‐induced PIP2 response. Using non‐invasive microelectrode ion flux estimation, we discovered that the Spm‐triggered K+ efflux response was strongly reduced in pip5k7 pip5k9 seedlings. Together, our results provide biochemical and genetic evidence for a physiological role of PIP2 in polyamine‐mediated signalling controlling K+ flux in plants.

Effects of insulin on IGF-1 receptors in equine lamellar tissue in vitro

Although it is understood that equine endocrinopathic laminitis can be triggered by high concentrations of insulin, it is unclear whether this represents a direct action on lamellar tissue via insulin receptors (InsR), an interaction with IGF-1 receptors (IGF-1R), or some other, indirect action. This uncertainty is because of the reported scarcity of InsR in lamellar tissue and the low affinity of insulin for equine IGF-1R. In the present study, the effects of insulin and IGF-1 (as a positive control) were examined using lamellar explants isolated from the hooves of healthy horses and incubated in cell culture medium for between 2 min and 48 h. In this system, a low physiological concentration of IGF-1 (10 nM; 1.31 ng/mL) caused a marked increase in the appearance of phosphorylated IGF-1R after 5 min (P < 0.05), and this effect was blocked by a human anti-IGF-1R monoclonal antibody (mAb). However, a high concentration of insulin (10 nM; 1,430 μIU/mL) appeared to cause dephosphorylation of the IGF-1R after 5 min (P < 0.01), 15 min, and 30 min (P < 0.001). Using 3H-thymidine as a marker, it was also demonstrated that insulin and IGF-1–stimulated cell proliferation in lamellar explants over the same concentration range as each other (1–100 nM), implying that each peptide acts via its own receptor (P < 0.001). Conversely, the effect of both peptides could be blocked using a selective anti-IGF-1R mAb (P < 0.001), implying that insulin acts via IGF1-R (either directly or indirectly). Notwithstanding this conundrum, the results demonstrate that insulin acts directly on lamellar tissue and suggest that a therapeutic anti-IGF-1R mAb could be useful in treating or preventing endocrinopathic laminitis.

UHPLC-MS/MS profiling of histidine and bile acid metabolism in human gastric fluid for diagnosis of gastric diseases

Bile acids (BAs) are synthesized in the liver and can mediate homeostasis and various metabolism processes in the human body. Their levels in the gastrointestinal tract are closely related to various gastrointestinal diseases. In particular, farnesoid X receptor activated by free BAs is associated with overexpression of histidine decarboxylase in tumorigenesis. Therefore, comprehensive profiling of histamine (HIST), histidine (His), and BAs in biological samples can provide insight into the pathological mechanisms of gastrointestinal diseases. However, development of an analytical platform to profile HIST, His, and BAs in biological samples has several challenges such as highly different polarities between acidic and basic targets, low physiological concentrations of analytes, and high matrix interference of biological samples. In this study, an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method combined with serial derivatization was developed to simultaneously determine HIST, His, and 5 BAs (cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, and lithocholic acid) in human gastric fluid. In serial derivatization, benzoyl chloride (BzCl) and N,N-dimethylethylenediamine (DMED) were used to selectively derivatize amino and carboxyl groups of analytes, respectively. After serial derivatization, all target derivatives were determined using a reverse-phase C18 LC column and positive multiple reaction monitoring (MRM) mode, with reasonable chromatographic separation and sensitive MS detection. To accurately quantify target metabolites, 7 stable isotope-labeled internal standards were used. The MS/MS spectra of DMED and Bz derivatives exhibited specific fragments via loss of a neutral molecule (dimethylamine; 45 Da) and inductive cleavage (benzoyl; m/z 105) from protonated molecules, enabling selection of appropriate MRM transition ions for selective and sensitive detection. The developed method was validated with respect to limits of detection and quantification, linearity, precision, accuracy, stability, and matrix effect. The established method was successfully applied to human gastric fluid samples. This method provides reliable quantification of HIST, His, and BAs in human gastric fluid and will be helpful to understand pathophysiological mechanisms of gastric diseases.

P1100IMPACT OF THE RELATIONSHIP BETWEEN LOW MOLECULAR WEIGHT PROTEINS REMOVAL EFFICIENCIES AND THE VOLUME OF ALBUMIN LEAKAGE IN HEMODIAFILTRATION THERAPY

Abstract Background and Aims The purpose of hemodiafiltration (HDF) is to remove small- to large molecular weight solutes in order to prevent and treat complications and to improve the prognosis of dialysis patients. Especially, larger low molecular weight proteins (LMWPs) must be removed efficiently. β2-microglobulin (MG) (molecular weight [MW], 11.8 kDa: Stokes radius, 15.6A), is considered as an important marker of dialysis efficiency. However, MW of β2-MG is too small as a marker in HDF. Conversely, α1-MG (MW, 33 kDa: Stokes radius, 28.4A) is the most appropriate marker for evaluation of solute removal efficiency of HDF because of the following reasons: (1) optical molecular size; (2) not very low physiological blood concentration; (3) stable production rate; (4) removal by convection; and (5) accumulation in ESRD. Efficient removal of α1-MG without losing albumin (Alb) (MW,66 kDa: Stokes radius, 35.5A) is impossible even by HDF. In this study, we investigated the relationship between LMWP removal efficiencies and the volume of Alb leakage by analyzing the results of patients undergoing HDF. And we also evaluated the trend of the serum levels of patients who had received high-efficiency HDF for more than three years Method Eighty-seven patients who had received HDF in 2018–2019 were included. The results of HDF (a total of 435 sessions) were analyzed (blood flow rate, 261.5±28.5 mL/min; replacement fluid volume, 48.7 L/s pre-dilution on-line HDF and 14.2 L/s post-dilution on-line HDF). The removal efficiencies of β2-MG, prolactin (PRL), and α1-MG, and the volume of Alb leakage were examined. In addition, the relationship between the removal efficiency of each solute and the volume of Alb leakage was investigated. Changes in serum Alb level were analyzed in 13 patients who continued to receive high-efficiency HDF (the removal rate of α1-MG was 30% or more) for more than three years. Results Analysis of the results (mean ± SD) obtained from a total of 435 sessions (87 patients) showed that the removal rates of β2-MG, PRL, and α1-MG were 80.7 ± 4.5%, 75.8±9.4 and 33.8 ± 9.4%, respectively, and the Alb leakage was 3.9 ± 1.8 g/s. The equations of the approximate curve between Alb leakage and β2-MG, PRL, and α1-MG removal rates were expressed as follows: ï½™= 1.4197ln(x) + 78.972 (R2 = 0.0426), y = 9.7185ln(x) + 64.134 (R2 =0.4632), and ï½™™= 11.225ln(x) + 20.282 (R2 = 0.6191), respectively. These results indicate that the dynamics between Alb leakage and α1-MG removal are very similar. That is, although the molecular weight of Alb is twice as that of α1-MG, the difference in their Stokes radius is 20%; therefore, separate removal of Alb and α1-MG is impossible. The mean serum Alb level in 13 patients who received high-efficiency HDF for 3 years or longer was 3.59 ± 0.23 g/dL in January 2017 and 3.69 ± 0.28 g/dL in December 2019, revealing no changes. Conclusion The efficient removal of larger LMWPs leads to the leakage of Alb. However, an Alb leakage of 3 to 6 g/s has little influence on the serum Alb level of patients. Thus, α1-MG is the most appropriate marker of the solute removal efficiency of HDF.

Antioxidant lipoic acid ligand-shell gold nanoconjugates against oxidative stress caused by α-synuclein aggregates.

Gold nanoparticles are becoming a promising platform for the delivery of drugs to treat neurodegenerative diseases. Parkinson's disease, associated with the aggregation of α-synuclein, is a condition that results in dysfunctional neuronal cells leading to their degeneration and death. Oxidative stress has been strongly implicated as a common feature in this process. The limited efficacy of the traditional therapies and the development of associated severe side effects present an unmet need for preventive and adjuvant therapies. The organosulfur compound lipoic acid, naturally located in the mitochondria, plays a powerful antioxidative role against oxidative stress. However, the efficacy is limited by its low physiological concentration, and the administration is affected by its short half-life and bioavailability due to hepatic degradation. Here we exploited the drug delivery potential of gold nanoparticles to assemble lipoic acid, and administered the system into SH-SY5Y cells, a cellular model commonly used to study Parkinson's disease. We tested the nanoconjugates of GNPs–LA, under an oxidative environment induced by gold nanoparticle/α-synuclein conjugates (GNPs–α-Syn). GNPs–LA were found to be biocompatible and capable of restoring the cell damage caused by high-level reactive oxygen species generated by excessive oxidative stress in the cellular environment. We conclude that GNPs–LA may serve as promising drug delivery vehicles conveying antioxidant molecules for the treatment of Parkinson's disease.

A targeted neurotransmitter quantification and nontargeted metabolic profiling method for pharmacometabolomics analysis of olanzapine by using UPLC-HRMS.

Neurotransmitters (NTs) are specific endogenous metabolites that act as “messengers” in synaptic transmission and are widely distributed in the central nervous system. Olanzapine (OLZ), a first-line antipsychotic drug, plays a key role in sedation and hypnosis, but, it presents clinical problems with a narrow therapeutic window, large individual differences and serious adverse effects, as well as an unclear mechanism in vivo. Herein, a simultaneous targeted NT quantification and nontargeted metabolomics method was developed and validated for pharmacometabolomics analysis of OLZ by using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS). Considering the low physiological concentrations of NTs, a full MS scan and target selective ion monitoring (tSIM) scan were combined for nontargeted metabolomics and targeted NT quantification, respectively. By using this strategy, NTs at a very low physiological concentration can be accurately detected and quantified in biological samples by tSIM scans. Moreover, simultaneously nontargeted profiling was also achieved by the full MS scan. The newly established UPLC-HRMS method was further used for the pharmacometabolomics study of OLZ. Statistical analysis revealed that tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, γ-aminobutyric acid etc. were significantly downregulated, while tyrosine was significantly upregulated, which suggested that OLZ could promote the downstream phase II reaction of 5-hydroxytryptamine, inhibit tyrosine hydroxylase activity, and increase the activity of γ-aminobutyric acid transaminase. In conclusion, this method could provide novel insights for revealing the pharmacodynamic effect and mechanism of antipsychotic drugs.

Number of subjects required in common study designs for functional GABA magnetic resonance spectroscopy in the human brain at 3 Tesla.

Magnetic resonance spectroscopy (MRS) is a research tool for measuring the concentration of metabolites such as γ‐aminobutyric acid (GABA) and glutamate in the brain. MEGA‐PRESS has been the preferred pulse sequence for GABA measurements due to low physiological GABA concentrations, hence low signal. To compensate, researchers incorporate long acquisition durations (7–10 min) making functional measurements of this metabolite challenging. Here, the acquisition duration and sample sizes required to detect specific concentration changes in GABA using MEGA‐PRESS at 3 T are presented for both between‐groups and within‐session study designs. 75 spectra were acquired during rest using MEGA‐PRESS from 41 healthy volunteers in 6 different brain regions at 3 T with voxel sizes between 13 and 22 cm3. Between‐group and within‐session variance was calculated for different acquisition durations and power calculations were performed to determine the number of subjects required to detect a given percentage change in GABA/NAA signal ratio. Within‐subject variability was assessed by sampling different segments of a single acquisition. Power calculations suggest that detecting a 15% change in GABA using a 2 min acquisition and a 27 cm3 voxel size, depending on the region, requires between 8 and 93 subjects using a within‐session design. A between‐group design typically requires more participants to detect the same difference. In brain regions with suboptimal shimming, the subject numbers can be up to 4‐fold more. Collecting data for longer than 4 min in brain regions examined in this study is deemed unnecessary, as variance in the signal did not reduce further for longer durations.

Targeted Metabolomics of Plant Hormones and Redox Metabolites in Stomatal Immunity.

Phytohormones and redox metabolites are important molecules in a number of biological processes related to plant growth, development, and stress responses. Understanding how these metabolites are involved in abiotic and biotic stress is a frequent topic of plant biology research. However, many factors, such as low physiological concentrations and the inherent complexity of plant samples, make identification and quantification of these important metabolites difficult. Here, we describe a method for metabolite extraction from whole leaves and guard cell-enriched samples and a targeted metabolomics strategy for the identification and quantification of specific hormone- and redox-related metabolites. Inour experiment, we used the reference plant Arabidopsis thaliana infected with the biotrophic pathogen Pseudomonas syringe pv. tomato (Pst) DC3000, and examined the changes in hormone and redox metabolites in systemic leaves, using the targeted metabolomics strategy in order to investigate potential functions of these metabolites in systemic acquired resistance (SAR) during a plant's immune responses. The methods reported here can be expanded to other metabolites and other biological systems beyond plants and bacterial pathogens.

Structure-functional changes in eNAMPT at high concentrations mediate mouse and human beta cell dysfunction in type 2 diabetes

Aims/hypothesisProgressive decline in functional beta cell mass is central to the development of type 2 diabetes. Elevated serum levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) are associated with beta cell failure in type 2 diabetes and eNAMPT immuno-neutralisation improves glucose tolerance in mouse models of diabetes. Despite this, the effects of eNAMPT on functional beta cell mass are poorly elucidated, with some studies having separately reported beta cell-protective effects of eNAMPT. eNAMPT exists in structurally and functionally distinct monomeric and dimeric forms. Dimerisation is essential for the NAD-biosynthetic capacity of NAMPT. Monomeric eNAMPT does not possess NAD-biosynthetic capacity and may exert distinct NAD-independent effects. This study aimed to fully characterise the structure-functional effects of eNAMPT on pancreatic beta cell functional mass and to relate these to beta cell failure in type 2 diabetes.MethodsCD-1 mice and serum from obese humans who were without diabetes, with impaired fasting glucose (IFG) or with type 2 diabetes (from the Body Fat, Surgery and Hormone [BodyFatS&H] study) or with or at risk of developing type 2 diabetes (from the VaSera trial) were used in this study. We generated recombinant wild-type and monomeric eNAMPT to explore the effects of eNAMPT on functional beta cell mass in isolated mouse and human islets. Beta cell function was determined by static and dynamic insulin secretion and intracellular calcium microfluorimetry. NAD-biosynthetic capacity of eNAMPT was assessed by colorimetric and fluorescent assays and by native mass spectrometry. Islet cell number was determined by immunohistochemical staining for insulin, glucagon and somatostatin, with islet apoptosis determined by caspase 3/7 activity. Markers of inflammation and beta cell identity were determined by quantitative reverse transcription PCR. Total, monomeric and dimeric eNAMPT and nicotinamide mononucleotide (NMN) were evaluated by ELISA, western blot and fluorometric assay using serum from non-diabetic, glucose intolerant and type 2 diabetic individuals.ResultseNAMPT exerts bimodal and concentration- and structure-functional-dependent effects on beta cell functional mass. At low physiological concentrations (~1 ng/ml), as seen in serum from humans without diabetes, eNAMPT enhances beta cell function through NAD-dependent mechanisms, consistent with eNAMPT being present as a dimer. However, as eNAMPT concentrations rise to ~5 ng/ml, as in type 2 diabetes, eNAMPT begins to adopt a monomeric form and mediates beta cell dysfunction, reduced beta cell identity and number, increased alpha cell number and increased apoptosis, through NAD-independent proinflammatory mechanisms.Conclusions/interpretationWe have characterised a novel mechanism of beta cell dysfunction in type 2 diabetes. At low physiological levels, eNAMPT exists in dimer form and maintains beta cell function and identity through NAD-dependent mechanisms. However, as eNAMPT levels rise, as in type 2 diabetes, structure-functional changes occur resulting in marked elevation of monomeric eNAMPT, which induces a diabetic phenotype in pancreatic islets. Strategies to selectively target monomeric eNAMPT could represent promising therapeutic strategies for the treatment of type 2 diabetes.

A Novel Possible Role for Met Hemoglobin as Carrier of Hydrogen Sulfide in the Blood.

Significance: Along with other gasotransmitters nitric oxide (NO) and carbon monoxide, hydrogen sulfide (H2S) has recently emerged as an important signaling molecule with a particularly complex metabolism. Endogenous H2S reacts with multiple cellular targets, including protein ferric heme groups, to elicit physiological responses, such as regulation of local blood flow. Recent Advances: Recent in vitro evidence suggests that H2S at low physiological concentrations is carried in the blood as bound to the small fraction of oxidized ferric hemoglobin (metHb). A relatively stable metHb-sulfide complex forms when H2S and purified metHb react in vitro, with an affinity within the in vivo physiological range of sulfide in the blood. Formation and subsequent redox metabolism of metHb-sulfide complex have also been confirmed in isolated intact red blood cells (RBCs) containing enhanced metHb levels. Thus, H2S may function as an endocrine signaling molecule and elicit responses at sites away from the site of production. In addition, metHb, considered as an inert or pathological hemoglobin derivative, may have a novel potential physiological role in the transport of H2S in the blood. Critical Issues: The transport of H2S in the blood mediated by metHb would represent an O2-independent pH-dependent mechanism for the blood-mediated control of blood flow and as such it is critical to understand the in vivo significance of this transport. Future Directions: Major challenges must be resolved to understand how metHb may carry H2S in the RBCs, in particular determination of metHb-sulfide levels in the blood and identification of targets in the vasculature.

3-Hydroxykynurenine bound to eye lens proteins induces oxidative modifications in crystalline proteins through a type I photosensitizing mechanism

Photosensitized reactions mediated by endogenous chromophores have been associated with the etiology of age-related cataract disease. Endogenous chromophores such as 3-hydroxykynurenine (3OHKN) can be found in both free form, and bound to crystallin proteins. However, their efficiency in generating photo-induced oxidative modifications on eye lens proteins is not completely understood. In this work, the efficiency and photodynamic activity of 3OHKN bound to both lysine (3OHKN-Lys) and bovine lens proteins (3OHKN-BLP) was assessed and compared with the photosensitizing activity of the major chromophore arising from glucose degradation (GDC). The photosensitizing activity of 3OHKN-Lys, 3OHKN-BLP and GDC was characterized by measurement of singlet oxygen quantum yields, O2 consumption, SDS-PAGE and amino acid analysis of the photo-oxidized proteins.Singlet oxygen quantum yields under 20% O2 atmosphere were 0.02, 0.01, and 0.27 for 3OHKN-Lys, 3OHKN-BLP and GDC, respectively. O2 consumption by photosensitized reactions was more efficient for 3OHKN-BLP, with the extent of O2 consumption being ∼28% higher than for 3OHKN-Lys and GDC under both 5 and 20% O2. SDS-PAGE showed that protein crosslinking is dependent on the O2 concentration, and more extensive at 5 than 20% O2. GDC and 3OHKN-Lys were the most efficient crosslinkers at 20 and 5% O2, respectively. Amino acid analysis of the irradiated proteins showed consumption of Trp, His, Tyr and Phe, and formation of kynurenine (from Trp), methionine sulfoxide (from Met) and DOPA (from Tyr). Kynurenine formation was dependent on the O2 concentration with higher amounts detected at 5 than 20% O2 for 3OHKN-BLP and 3OHKN-Lys, with 3OHKN-BLP the most efficient sensitizer. Our results suggest that 3OHKN-BLP can elicit photo-oxidative damage mainly by a type I photosensitizing mechanism, with this likely to be the most prevalent pathway at the low physiologic O2 concentrations in the eye lens.

Effects of Complex Formation on Motion in DNA Binding Domains of ARFs

The auxin response factor (ARF) family is a set of DNA binding transcription factors found in the model plant Arabidopsis thaliana in addition to many other food and fuel crops. Along with two other frequently conserved domains, all ARFs contain an N‐terminus DNA binding domain (DBD) that includes a B3 DNA binding motif and a dimerization subdomain. While the structure of example DBDs have been solved, motion within this domain is poorly understood. The interplay of these domains may provide selectivity in binding to variants of target DNA constructs. Our work examines the DBDs of ARF1 and ARF5, a transcription repressor and transcription activator respectively, as ARF5 is known to be more permissive than ARF1 to a larger spacing between binding sites on DNA. Our work directly tests the hypothesis that the ability to bind to a broader range of DNA targets relates to observable differences in structural flexibility. We combine spectroscopic and chromatographic methods of resolving both complex formation and molecular motion for different combinations of target DNA and DBD. We utilize fluorescence spectroscopy as it resolves protein motions and associations that occur on the same timescale as the fluorescent lifetimes (~ns). Labeled proteins can also be studied at low physiological concentrations and within an aqueous buffer. We optimized the expression and purification of the protein as well as two alternative fluorophore conjugation methods to compare thiol‐reactive fluorophore labeling to a newer technique that targets the N‐terminus. Under conditions of known dissociated versus associated states, we apply both steady‐state and time‐resolved fluorescence anisotropy to sense rotational correlation times of these proteins and distinguish subdomain motions in the presence and absence of DNA. Our work demonstrates that the N‐terminal probes are more sensitive to molecular changes likely due to its placement at the hinge between the DNA binding domain and the dimerization domain. Consistent with our hypothesis, ARF5 shifts from a highly dynamic state to a more rigid state upon complex formation while ARF1 remains more unperturbed. These differences in the available structural ensemble provide valuable insights into the structural basis of target specificity as well as the interplay of these transcriptional on‐ and off‐switches.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Choroid plexus glutathione peroxidases are instrumental in protecting the brain fluid environment from hydroperoxides during postnatal development.

Hydrogen peroxide, released at low physiological concentration, is involved in different cell signaling pathways during brain development. When released at supraphysiological concentrations in brain fluids following an inflammatory, hypoxic, or toxic stress, it can initiate lipid peroxidation, protein, and nucleic acid damage and contribute to long-term neurological impairment associated with perinatal diseases. We found high glutathione peroxidase and glutathione reductase enzymatic activities in both lateral and fourth ventricle choroid plexus tissue isolated from developing rats, in comparison to the cerebral cortex and liver. Consistent with these, a high protein expression of glutathione peroxidases 1 and 4 was observed in choroid plexus epithelial cells, which form the blood-cerebrospinal fluid barrier. Live choroid plexuses isolated from newborn rats were highly efficient in detoxifying H2O2 from mock cerebrospinal fluid, illustrating the capacity of the choroid plexuses to control H2O2 concentration in the ventricular system of the brain. We used a differentiated cellular model of the blood-cerebrospinal fluid barrier coupled to kinetic and inhibition analyses to show that glutathione peroxidases are more potent than catalase to detoxify extracellular H2O2 at concentrations up to 250 µM. The choroidal cells also formed an enzymatic barrier preventing blood-borne hydroperoxides to reach the cerebrospinal fluid. These data point out the choroid plexuses as key structures in the control of hydroperoxide levels in the cerebral fluid environment during development, at a time when the protective glial cell network is still immature. Glutathione peroxidases are the main effectors of this choroidal hydroperoxide inactivation.