Synergistic effect of manganese (II) phosphate & diamond nanoparticles in electrochemical sensors for reactive oxygen species determination in seminal plasma.

In this work, we explore the ability of manganese (II) phosphate (MnP) as a catalytic element for the determination of reactive oxygen species (ROS) in seminal plasma, when MnP is employed as modifier of a glassy carbon electrode. The electrochemical response of the manganese (II) phosphate-modified electrode shows a wave at around +0.65 V due to the oxidation of Mn2+ to MnO2+, which is clearly enhanced after addition of superoxide, the molecule considered as the mother of ROS. Once proved the suitability of manganese (II) phosphate as catalyst, we evaluate the effect of including a 0D (diamond nanoparticles) or a 2D (ReS2) nanomaterial in the sensor design. The system consisting of manganese (II) phosphate and diamond nanoparticles yielded the largest improvement of the response. The morphological characterization of the sensor surface was performed by scanning electron microscopy and atomic force microscopy, while cyclic and differential pulse voltammetry were employed for the electrochemical characterization of the sensor. After optimizing the sensor construction, calibration procedures by chronoamperometry were performed, leading to a linear relation between peak intensity and the superoxide concentration in the range of 1.1 10-4 M - 1.0 10-3 M with a limit of detection of 3.2 10-5 M. Seminal plasma samples were analysed by the standard addition method. Moreover, the analysis of samples fortified with superoxide at the µM level leads to recoveries of 95%.

Lactate biosensing based on covalent immobilization of lactate oxidase onto chevron-like graphene nanoribbons via diazotization-coupling reaction.

We have designed and prepared an electrochemical biosensor for lactate determination. Through a diazotation process, the enzyme lactate oxidase (LOx) is anchored onto chevron-like graphene nanoribbons (GNR), previously synthesized by a solution-based chemical route, and used as modifiers of glassy carbon electrodes. In a first step, we have performed the grafting of a 4-carboxyphenyl film, by electrochemical reduction of the corresponding 4-carboxyphenyl diazonium salt, on the GNR-modified electrode surface. In this way, the carboxylic groups are exposed to the solution, enabling the covalent immobilization of the enzyme through the formation of an amide bond between these carboxylic groups and the amine groups of the enzyme. The biosensor design was optimized through the morphological and electrochemical characterization of each construction step by atomic force microscopy, scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy.The cyclic voltammetric response of the biosensor in a solution of hydroxymethylferrocene in presence of l-lactate evidenced a clear electrocatalytic effect powered by the specific design of the biosensing platform with LOx covalently attached to the GNR layer. From the calibration procedures employed for l-lactate determination, a linear concentration range of 3.4 · 10-5- 2.8 · 10-4 M and a detection limit of 11 µM were obtained, with relative errors and relative standard deviations less than 6.0% and 8.4%, respectively. The applicability of the biosensor was tested by determining lactate in apple juices, leading to results that are in good agreement with those obtained with a well-established enzymatic spectrophotometric assay kit.

Electrochemical Improvements Can Be Realized via Shortening the Length of Screen-Printed Electrochemical Platforms.

Screen-printed electrodes (SPEs) are ubiquitous within the field of electrochemistry and are commonplace within the arsenal of electrochemists. Their popularity stems from their reproducibility, versatility, and extremely low-cost production, allowing their utilization as single-shot electrodes and thus removing the need for tedious electrode pretreatments. Many SPE studies have explored changing the working electrode composition and/or size to benefit the researcher's specific applications. In this paper, we explore a critical parameter of SPEs that is often overlooked; namely, we explore changing the length of the SPE connections. We provide evidence of resistance changes through altering the connection length to the working electrode through theoretical calculations, multimeter measurements, and electrochemical impedance spectroscopy (EIS). We demonstrate that changing the physical length of SPE connections gives rise to more accurate heterogeneous electrode kinetics, which cannot be overcome simply through IR compensation. Significant improvements are observed when utilized as the basis of electrochemical sensing platforms for sodium nitrite, ß-nicotinamide adenine dinucleotide (NADH), and lead (II). This work has a significant impact upon the field of SPEs and highlights the need for researchers to characterize and define their specific electrode performance. Without such fundamental characterization as the length and resistance of the SPE used, direct comparisons between two different systems for similar applications are obsolete. We therefore suggest that, when using SPEs in the future, experimentalists report the length of the working electrode connection alongside the measured resistance (multimeter or EIS) to facilitate this standardization across the field.

A supramolecular hybrid sensor based on cucurbit[8]uril, 2D-molibdenum disulphide and diamond nanoparticles towards methyl viologen analysis.

We develop an electrochemical sensor by using 2D-transition metal dichalcogenides (TMD), specifically MoS2, and nanoparticles stabilized with cucurbit[8]uril (CB[8]) incorporated together with them. Two different nanoparticles are assayed: diamond nanoparticles (DNPs) and gold nanoparticles (AuNp). 0D materials, together with TMD, provide increased conductivity and active surface while the macrocycle CB[8] affords selectivity towards the guest methyl viologen (MV2+), also named paraquat. Glassy Carbon (GC) electrodes are modified by drop-casting of suspensions of MoS2, followed by either a CB[8]-DNPs hybrid dispersion or a CB[8]-AuNp suspension. Atomic force microscopy is employed for the morphological characterization of the electrochemical sensor surface while cyclic voltammetry and electrochemical impedance spectroscopy techniques allow the electrochemical characterization of the sensor. The well-stablished signals of CB[8]-encapsulated MV2+ arise in voltammetric measurements when the macrocycle modifies the 0D-materials. Once the sensor construction and differential pulse voltammetry parameters have been optimized for quantification purposes, calibration procedures are performed with the platform GC/MoS2/CB[8]-DNPs. This sensing platform shows linear relations between peak intensity and the MV2+ concentration in the linear concentration range of (0.73-8.0) · 10-6 M with a limit of detection of 2.2 · 10-7 M. Analyses of river water samples fortified with MV2+ at the µM level shows recoveries of 100% with RSD values of 6.4% (n = 3).

MoS2 quantum dots for on-line fluorescence determination of the food additive allura red.

This work presents an on-line fluorescence method for the allura red (AR) determination. The method is based on the fluorescence quenching of dots of MoS2 because of their interaction with the non-fluorescence dye. MoS2-dots were synthetized and characterized by spectroscopic techniques and High Resolution Transmission Electronic Microscopy (HR-TEM). The simultaneous injection of the nanomaterial and the dye in a flow injection analysis system allows the determination of allura red at 1.7 × 10-6 M concentration level with a very good accuracy and precision (Er minor than 10% and RSD lower than 8%) and a sampling frequency of 180 samples per hour. Moreover, the interaction fluorophore-quencher results a dynamic inhibition mechanism. The proposed methodology was applied to the AR analysis in soft beverages and powders for gelatine preparation. Colourant concentrations of 63 ± 2 mg/L (n = 3) and 0.30 ± 0.01 mg/g (n = 3) were found, respectively. These results were validated by high performance liquid chromatography technique.

Arsenic trioxide-increased MDCK cells proliferation requires activator protein 1-mediated increase of the sodium/proton exchanger 1 activity.

The release of protons (H+) occurs via the Na+/H+ exchanger isoform 1 (NHE1) leading to a stable intracellular pH (pHi) in MDCK cells. Chronic intake of arsenic trioxide (ATO), in the drinking water, associated with higher morbidity and mortality in neoplastic tissues. ATO increased NHE1 expression and activity, resulting in intracellular alkalization and higher MDCK cells proliferation. Since the pro-proliferative transcription factor activator protein 1 (AP-1) gets activated by al alkaline intracellular pH, a phenomenon paralleled by higher NHEs activity, we asked whether ATO-increased MDCK cells proliferation involves AP-1-dependent NHE1 activation. Cells were exposed (48 h) to ATO (0.05 µmol/L), SR11302 (1 µmol/L, AP-1 inhibitor), HOE-694 (100 nmol/L, NHE1 inhibitor) and EIPA (50 µmol/L, NHE1/NHE3 inhibitor) in the presence of S3226 (10 µmol/L, NHE3 inhibitor), concanamycin A (0.1 µmol/L, V-ATPases inhibitor), and Schering (10 µmol/L, H+/K+-ATPase inhibitor). [3H]Thymidine incorporation, cell counting, wound healing assay, and AP-1 activity were determined. The pHi was measured in cells pre-loaded (10 min) with 2,7-bicarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (12 mmol/L) and exposed to NH4Cl (20 mmol/L). Basal pHi and recovery rate (dpHi/dt), intracellular buffer capacity (ßi) and H+ flux (JH+) were determined. NHE1 protein abundance was measured by Western blotting and immunofluorescence. ATO increased the cell growth (1.5 fold), basal pHi (0.4 pHi units), dpHi/dt (1.8 fold), JH+ (1.4 fold), AP-1 activity and NHE1 protein abundance (1.3 fold). ATO also increased (1.5 fold) the nuclear/perinuclear NHE1 immunosignal. SR11302 and HOE-694 blocked ATO effects. Thus, ATO-increased proliferation resulted from AP-1-dependent NHE1 activation in MDCK cells.

Chemically synthesized chevron-like graphene nanoribbons for electrochemical sensors development: determination of epinephrine.

We employ chevron-like graphene nanoribbons (GNRs) synthesized by a solution-based chemical route to develop a novel electrochemical sensor for determination of the neurotransmitter epinephrine (EPI). The sensor surface, a glassy carbon electrode modified with GNRs, is characterized by atomic force microscopy, scanning electron microscopy and Raman spectroscopy, which show that the electrode surface modification comprises of bi-dimensional multilayer-stacked GNRs that retain their molecular structure. The charge transfer process occurring at the electrode interface is evaluated by electrochemical impedance spectroscopy. The sensor is applied to the determination of EPI, employing as an analytical signal the reduction peak corresponding to the epinephrinechrome-leucoepinephrinechrome transition (E = - 0.25 V) instead of the oxidation peak usually employed in the literature (E = + 0.6 V) in order to minimize interferences. The results obtained demonstrate that chevron-like nanoribbons synthesized by solution methods exhibit reliable electrocatalytic activity for EPI determination. Using differential pulse voltammetry, we obtain a linear concentration range from 6.4 × 10-6 to 1.0 × 10-4 M and a detection limit of 2.1 × 10-6 M. The applicability of the sensor was evaluated by determining EPI in pharmaceutical samples with satisfactory results.

Evaluation of oxidative stress: Nanoparticle-based electrochemical sensors for hydrogen peroxide determination in human semen samples.

We have developed electrochemical sensors for the determination of H2O2 in a complex matrix such as human semen as a method to evaluate oxidative stress related to male infertility. Our sensors are based on the modification of conventional electrode surfaces with nanoparticles. We used diamond nanoparticles (DNp) either on glassy carbon or gold surfaces (GC/DNp and Au/DNp sensors, respectively), and copper nanoparticles electrochemically generated directly on glassy carbon surfaces (GC/CuNp). The morphology of the modified electrode surfaces was characterized by Atomic Force Microscopy (AFM), and the H2O2 determination performance evaluated by chronoamperometric measurements at different applied potentials. The best results are obtained for GC/DNp at +1.0 V, Au/DNp at -0.6 V and GC/CuNp at +0.2 V with detection limits (LD) of 1.1 µM, 2.4 µM and 2.6 µM, respectively. The analysis of H2O2 in doped synthetic semen using the GC/CuNp sensor shows the best recoveries, reaching a mean value of 103%. The GC/CuNp sensor was successfully applied to H2O2 analysis in real human semen. In this case, a H2O2 concentration of 1.42 ± 0.05 mM is found and recoveries of 102% on average are obtained.

Fluorescence enhancement of fungicide thiabendazole by van der Waals interaction with transition metal dichalcogenide nanosheets for highly specific sensors.

Many molecules quench their fluorescence upon adsorption on surfaces. Herein we show that the interaction of thiabendazole, a widespread used fungicide of the benzimidazole family, with nanosheets of transition metal dichalcogenides, particularly of WS2, leads to a significant increase, more than a factor of 5, of the fluorescence yield. This surprising effect is rationalized by DFT calculations and found to be related to the inhibition of the intramolecular rotation between the benzimidazole and thiazole groups due to a bonding rigidization upon interaction with the MoS2 surface. This non-covalent adsorption leads to a redistribution of the molecular LUMO that blocks the non-radiative energy dissipation channel. This unusual behaviour does not operate either for other molecules of the same benzimidazole family or for other 2D materials (graphene or graphene oxide). Moreover, we found that a linear dependence of the emission with the concentration of thiabendazole in solution, which combined with the specificity of the process, allows the development of a highly sensitive and selective method towards thiabendazole determination that can be applied to real river water samples. An excellent detection limit of 2.7 nM, comparable to the best performing reported methods, is obtained with very good accuracy (Er ≤ 6.1%) and reproducibility (RSD ≤ 4.1%) in the concentration range assayed.

Differential pulse voltammetric determination of the carcinogenic diamine 4,4'-oxydianiline by electrochemical preconcentration on a MoS2 based sensor.

An electrochemical sensor for the carcinogen 4,4'-oxydianiline (Oxy) is described. The method is based on the ability of MoS2 nanosheets to preconcentrate Oxy. A glassy carbon electrode (GCE) was covered, by drop-casting, with MoS2 nanosheets that were obtained by exfoliation. X-Ray photoemission spectroscopy indicates that Oxy accumulates on the MoS2 nanosheets through an electropolymerization process similar to that reported for aniline. Both electrochemical impedance spectroscopy and atomic force microscopy were used to characterize the electrode surface at the different stages of device fabrication. Employing the current measured at +0.27 V vs. Ag/AgCl after Oxy adsorption, the modified GCE enables the voltammetric detection of Oxy at 80 nM levels with relative errors and relative standard deviations of <8.3 and <5.6%, respectively, at all the concentrations studied. The method was applied to the selective determination of Oxy in spiked river water samples. Very good selectivity and recoveries of around 95% in average are found. Graphical abstractSchematic representation of 4,4-oxydianiline electrochemical polymerization and preconcentration onto molybdenum disulfide nanosheets for the diamine determination in river waters.

Intracellular acidification reduces l-arginine transport via system y+L but not via system y+/CATs and nitric oxide synthase activity in human umbilical vein endothelial cells.

l-Arginine is taken up via the cationic amino acid transporters (system y+/CATs) and system y+L in human umbilical vein endothelial cells (HUVECs). l-Arginine is the substrate for endothelial NO synthase (eNOS) which is activated by intracellular alkalization, but nothing is known regarding modulation of system y+/CATs and system y+L activity, and eNOS activity by the pHi in HUVECs. We studied whether an acidic pHi modulates l-arginine transport and eNOS activity in HUVECs. Cells loaded with a pH-sensitive probe were subjected to 0.1-20 mmol/L NH4Cl pulse assay to generate pHi 7.13-6.55. Before pHi started to recover, l-arginine transport (0-20 or 0-1000 µmol/L, 10 s, 37 °C) in the absence or presence of 200 µmol/L N-ethylmaleimide (NEM) (system y+/CATs inhibitor) or 2 mmol/L l-leucine (systemy+L substrate) was measured. Protein abundance for eNOS and serine1177 or threonine495 phosphorylated eNOS was determined. The results show that intracellular acidification reduced system y+L but not system y+/CATs mediated l-arginine maximal transport capacity due to reduced maximal velocity. Acidic pHi reduced NO synthesis and eNOS serine1177 phosphorylation. Thus, system y+L activity is downregulated by an acidic pHi, a phenomenon that may result in reduced NO synthesis in HUVECs.

Can solvent induced surface modifications applied to screen-printed platforms enhance their electroanalytical performance?

In this paper the effect of solvent induced chemical surface enhancements upon graphitic screen-printed electrodes (SPEs) is explored. Previous literature has indicated that treating the working electrode of a SPE with the solvent N,N-dimethylformamide (DMF) offers improvements within the electroanalytical response, resulting in a 57-fold increment in the electrode surface area compared to their unmodified counterparts. The protocol involves two steps: (i) the SPE is placed into DMF for a selected time, and (ii) it is cured in an oven at a selected time and temperature. Beneficial electroanalytical outputs are reported to be due to the increased surface area attributed to the binder within the bulk surface of the SPEs dissolving out during the immersion step (step i). We revisit this exciting concept and explore these solvent induced chemical surface enhancements using edge- and basal-plane like SPEs and a new bespoke SPE, utilising the solvent DMF and explore, in detail, the parameters utilised in steps (i) and (ii). The electrochemical performance following steps (i) and (ii) is evaluated using the outer-sphere redox probe hexaammineruthenium(iii) chloride/0.1 M KCl, where it is found that the largest improvement is obtained using DMF with an immersion time of 10 minutes and a curing time of 30 minutes at 100 °C. Solvent induced chemical surface enhancement upon the electrochemical performance of SPEs is also benchmarked in terms of their electroanalytical sensing of NADH (dihydronicotinamide adenine dinucleotide reduced form) and capsaicin both of which are compared to their unmodified SPE counterparts. In both cases, it is apparent that a marginal improvement in the electroanalytical sensitivity (i.e. gradient of calibration plots) of 1.08-fold and 1.38-fold are found respectively. Returning to the original exciting concept, interestingly it was found that when a poor experimental technique was employed, only then significant increases within the working electrode area are evident. In this case, the insulating layer that defines the working electrode surface, which was not protected from the solvent (step (i)) creates cracks within the insulating layer exposing the underlying carbon connections and thus increasing the electrode area by an unknown quantity. We infer that the origin of the response reported within the literature, where an extreme increase in the electrochemical surface area (57-fold) was reported, is unlikely to be solely due to the binder dissolving but rather poor experimental control over step (i).

Revisiting PC1/3 Mutants: Dominant-Negative Effect of Endoplasmic Reticulum-Retained Mutants.

Prohormone convertase 1/3 (PC1/3), encoded by the gene PCSK1, is critical for peptide hormone synthesis. An increasing number of studies have shown that inactivating mutations in PCSK1 are correlated with endocrine pathologies ranging from intestinal dysfunction to morbid obesity, whereas the common nonsynonymous polymorphisms rs6232 (N221D) and rs6234-rs6235 (Q665E-S690T) are highly associated with obesity risk. In this report, we revisited the biochemical and cellular properties of PC1/3 variants in the context of a wild-type PC1/3 background instead of the S357G hypermorph background used for all previous studies. In the wild-type background the PC1/3 N221D variant exhibited 30% lower enzymatic activity in a fluorogenic assay than wild-type PC1/3; this inhibition was greater than that detected in an equivalent experiment using the PC1/3 S357G background. A PC1/3 variant with the linked carboxyl-terminal polymorphisms Q665E-S690T did not show this difference. We also analyzed the biochemical properties of 2 PC1/3 mutants, G209R and G593R, which are retained in the endoplasmic reticulum (ER), and studied their effects on wild-type PC1/3. The expression of ER-retained mutants induced ER stress markers and also resulted in dominant-negative blockade of wild-type PC1/3 prodomain cleavage and decreased expression of wild-type PC1/3, suggesting facilitation of the entry of wild-type protein to a degradative proteasomal pathway. Dominant-negative effects of PC1/3 mutations on the expression and maturation of wild-type protein, with consequential effects on PC1/3 availability, add a new element which must be considered in population and clinical studies of this gene.

Identification of potent and compartment-selective small molecule furin inhibitors using cell-based assays.

The proprotein convertase furin is implicated in a variety of pathogenic processes such as bacterial toxin activation, viral propagation, and cancer. Several groups have identified non-peptide compounds with high inhibitory potency against furin in vitro, although their efficacy in various cell-based assays is largely unknown. In this study we show that certain guanidinylated 2,5-dideoxystreptamine derivatives exhibit interesting ex vivo properties. Compound 1b (1,1'-(4-((2,4-diguanidino-5-(4-guanidinophenoxy)cyclohexyl)oxy)-1,3-phenylene)diguanidine) is a potent and cell-permeable inhibitor of cellular furin, since it was able to retard tumor cell migration, block release of a Golgi reporter, and protect cells against Bacillus anthracis (anthrax) and Pseudomonas aeruginosa intoxication, with no evident cell toxicity. Other compounds based on the 2,5-dideoxystreptamine scaffold, such as compound 1g (1,1'-(4,6-bis(4-guanidinophenoxy)cyclohexane-1,3-diyl)diguanidine) also efficiently protected cells against anthrax, but displayed only moderate protection against Pseudomonas exotoxin A and did not inhibit cell migration, suggesting poor cell permeability. Certain bis-guanidinophenyl ether derivatives such as 2f (1,3-bis(2,4-diguanidinophenoxy) benzene) exhibited micromolar potency against furin in vitro, low cell toxicity, and highly efficient protection against anthrax toxin; this compound only slightly inhibited intracellular furin. Thus, compounds 1g and 2f both represent potent furin inhibitors at the cell surface with low intracellular inhibitory action, and these particular compounds might therefore be of preferred therapeutic interest in the treatment of certain bacterial and viral infections.

A novel familial mutation in the PCSK1 gene that alters the oxyanion hole residue of proprotein convertase 1/3 and impairs its enzymatic activity.

Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, encoding the neuroendocrine convertase 1 precursor (PC1/3) which was recently reported as a cause of Congenital Diarrhea Disorder (CDD). The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that this variant accounts for the enteric and systemic endocrinopathies seen in this large consanguineous kindred.

Biochemical and cell biological properties of the human prohormone convertase 1/3 Ser357Gly mutation: a PC1/3 hypermorph.

Satiety and appetite signaling are accomplished by circulating peptide hormones. These peptide hormones require processing from larger precursors to become bioactive, often by the proprotein convertase 1/3 (PC1/3). Several subcellular maturation steps are necessary for PC1/3 to achieve its optimal enzymatic activity. Certain PC1/3 variants found in the general population slightly attenuate its enzymatic activity and are associated with obesity and diabetes. However, mutations that increase PC1/3 activity and/or affect its specificity could also have physiological consequences. We here present data showing that the known human Ser357Gly PC1/3 mutant (PC1/3(S357G)) represents a PC1/3 hypermorph. Conditioned media from human embryonic kidney-293 cells transfected with PC1/3(WT) and PC1/3(S357G) were collected and enzymatic activity characterized. PC1/3(S357G) exhibited a lower calcium dependence; a higher pH optimum (neutral); and a higher resistance to peptide inhibitors than the wild-type enzyme. PC1/3(S357G) exhibited increased cleavage to the C-terminally truncated form, and kinetic parameters of the full-length and truncated mutant enzymes were also altered. Lastly, the S357G mutation broadened the specificity of the enzyme; we detected PC2-like specificity on the substrate proCART, the precursor of the cocaine- and amphetamine regulated transcript neuropeptide known to be associated with obesity. The production of another anorexigenic peptide normally synthesized only by PC2, αMSH, was increased when proopiomelanocortin was coexpressed with PC1/3(S357G). Considering the aberrant enzymatic profile of PC1/3(S357G), we hypothesize that this enzyme possesses unusual processing activity that may significantly change the profile of circulating peptide hormones.

Defective transport of the obesity mutant PC1/3 N222D contributes to loss of function.

Mutations in the PCSK1 gene encoding prohormone convertase 1/3 (PC1/3) are strongly associated with obesity in humans. The PC1/3(N222D) mutant mouse thus far represents the only mouse model that mimics the PC1/3 obesity phenotype in humans. The present investigation addresses the cell biology of the N222D mutation. Metabolic labeling experiments reveal a clear defect in the kinetics of insulin biosynthesis in islets from PC1/3(N222D) mutant mice, resulting in an increase in both proinsulin and its processing intermediates, predominantly lacking cleavage at the Arg-Arg site. Although the mutant PC1/3 zymogen is correctly processed to the 87-kDa form, pulse-chase immunoprecipitation experiments, labeling, and immunohistochemical experiments using uncleavable variants all demonstrate that the PC1/3-N222D protein is largely mislocalized compared with similar wild-type (WT) constructs, being predominantly retained in the endoplasmic reticulum. The PC1/3-N222D mutant also undergoes more efficient degradation via the ubiquitin-proteasome system than the WT enzyme. Lastly, the mutant PC1/3-N222D protein coimmunoprecipitates with WT PC1/3 and exerts a modest effect on intracellular retention of the WT enzyme. These profound alterations in the cell biology of PC1/3-N222D are likely to contribute to the defective insulin biosynthetic events observed in the mutant mice and may be relevant to the dramatic contributions of polymorphisms in this gene to human obesity.

Withdrawal from chronic amphetamine reduces dopamine transmission in the rat lateral septum.

The lateral septum (LS) is a brain nucleus implicated in the addictive process. This study investigated whether withdrawal from chronic amphetamine (AMPH) induces alterations in dopamine (DA) transmission within the LS. Male Sprague-Dawley rats were injected with AMPH (2.5 mg/kg i.p.) or saline during 14 days and thereafter subjected to 24 hr or 14 days of withdrawal. After these withdrawal periods, we measured DA extracellular levels by in vivo microdialysis, DA tissue levels, and tyrosine hydroxylase (TH) and vesicular monoamine transporter-2 (VMAT2) expression in the LS. Our results showed a significant decrease in K(+) -induced release of DA in the LS of AMPH-treated rats, 14 days after withdrawal compared with saline-treated rats. There were no significant differences in DA tissue content and TH expression. Interestingly, there was a decrease of LS VMAT2 expression in AMPH-treated rats, 14 days after withdrawal compared with saline-treated rats. This is the first neurochemical evidence showing that withdrawal from repeated AMPH administration decreases K(+) -induced DA release in the rat LS. Our results suggest that this decrease in DA releasability could be due to a decrease in DA vesicular uptake. The possibility that these neurochemical changes are associated with AMPH abstinence syndrome should be further explored.

An amphipathic alpha-helix in the prodomain of cocaine and amphetamine regulated transcript peptide precursor serves as its sorting signal to the regulated secretory pathway.

Cocaine and Amphetamine Regulated Transcript (CART) peptides are anorexigenic neuropeptides. The L34F mutation in human CART peptide precursor (proCART) has been linked to obesity (Yanik et al. Endocrinology 147: 39, 2006). Decrease in CART peptide levels in individuals carrying the L34F mutation was attributed to proCART subcellular missorting. We studied proCART features required to enter the regulated secretory pathway. The subcellular localization and the secretion mode of monomeric EGFP fused to the full-length or truncated forms of human proCART transiently transfected in PC12 cells were analyzed. Our results showed that the N-terminal 1-41 fragment of proCART was necessary and sufficient to sort proCART to the regulated secretory pathway. In silico modeling predicted an alpha-helix structure located between residues 24-37 of proCART. Helical wheel projection of proCART alpha-helix showed an amphipathic configuration. The L34F mutation does not modify the amphipathicity of proCART alpha-helix and consistently proCARTL34F was efficiently sorted to the regulated secretory pathway. However, four additional mutations to proCARTL34F that reduced its alpha-helix amphipathicity resulted in the missorting of the mutated proCART toward the constitutive secretory pathway. These findings show that an amphipathic alpha-helix is a key cis-structure for the proCART sorting mechanism. In addition, our results indicate that the association between L34F mutation and obesity is not explained by proCART missorting.

New supramolecular interactions for electrochemical sensors development: different cucurbit[8]uril sensing platform designs.

Three different strategies for cucurbit[8]uril immobilization on a glassy carbon electrode have been assayed. The electrochemical properties of the resulting modified electrodes in solutions containing neutral, positively and negatively charged potential cucurbit[8]uril guests were investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The comparison of the electrochemical behaviour exhibited by the unmodified electrodes against various probes, with respect to that of each modified electrode, resulted in an appropriate method to choose among different strategies for the development of electrochemical sensors. These sensors are based on the incorporation of the cucurbit[8]uril molecular selection properties that depend on the chemical characteristics of the potential analytes. Furthermore, atomic force microscopy was employed for the characterization of the different surfaces developed.