Redox Substrates Research Articles

Piperazine-Fused Cyclic Disulfides Unlock High-Performance Bioreductive Probes of Thioredoxins and Bifunctional Reagents for Thiol Redox Biology.

We report piperazine-fused six-membered-cyclic disulfides as redox substrates that unlock best-in-class bioreduction probes for live cell biology, since their self-immolation after reduction is unprecedentedly rapid. We develop scalable, diastereomerically pure, six-step syntheses that access four key cis- and trans-piperazine-fused cyclic dichalcogenides without chromatography. Fluorogenic redox probes using the disulfide piperazines are activated >100-fold faster than the prior art monoamines, allowing us to deconvolute reduction and cyclization rates during activation. The cis- and trans-fused diastereomers have remarkably different reductant specificities, which we trace back to piperazine boat/chair conformation effects: the cis-fused disulfide C-DiThia is activated only by strong vicinal dithiol reductants, but the trans-disulfide T-DiThia is activated even by moderate concentrations of monothiols such as GSH. Thus, in cellular applications, cis-disulfide probes selectively report on the reductive activity of the powerful thioredoxin proteins, while trans-disulfides are rapidly but promiscuously reactive. Finally, we showcase late-stage diversifications of the piperazine-disulfides, promising their broad applicability as redox-cleavable cores for probes and prodrugs that interface powerfully with cellular thiol/disulfide redox biology, for solid phase synthesis and purification, and for stimulus-responsive linkers in bifunctional reagents and antibody-drug conjugates - in addition to their dithiols' potential as high-performance reducing agents.

Electrochemical Characterization of Two Gut Microbial Strains Cooperatively Promoting Multiple Sclerosis Pathogenesis.

In this study, we explored the extracellular electron transfer (EET) capabilities of two bacterial strains, OTU0001 and OTU0002, which are demonstrated in biofilm formation in mouse gut and the induction of autoimmune diseases like multiple sclerosis. OTU0002 displayed significant electrogenic behaviour, producing microbial current on an indium tin-doped oxide electrode surface, particularly in the presence of glucose, with a current density of 60 nA/cm2. The presence of cell-surface redox substrate potentially mediating EET was revealed by the redox-based staining method and electrochemical voltammetry assay. However, medium swapping analyses and the addition of flavins, a model redox mediator, suggest that the current production is dominated by soluble endogenous redox substrates in OTU0002. Given redox substrates were detected at the cell surface, the secreted redox molecule may interact with the cellular surface of OTU0002. In contrast to OTU0002, OTU0001 did not exhibit notable electrochemical activity, lacking cell-surface redox molecules. Further, the mixture of the two strains did not increase the current production from OTU0001, suggesting that OTU0001 does not support the EET mechanism of OTU0002. The present work revealed the coexistence of EET and non-EET capable pathogens in multi-species biofilm.

Self-Immolative Electrochemical Redox Substrates: Emerging Artificial Receptors in Sensing and Biosensing.

The development of artificial receptors has great significance in measurement science and technology. The need for a robust version of natural receptors is getting increased attention because the cost of natural receptors is still high along with storage difficulties. Aptamers, imprinted polymers, and nanozymes are some of the matured artificial receptors in analytical chemistry. Recently, a new direction has been discovered by organic chemists, who can synthesize robust, activity-based, self-immolative organic molecules that have artificial receptor properties for the targeted analytes. Specifically designed trigger moieties implant selectivity and sensitivity. These latent electrochemical redox substrates are highly stable, mass-producible, inexpensive, and eco-friendly. Combining redox substrates with the merits of electrochemical techniques is a good opportunity to establish a new direction in artificial receptors. This Review provides an overview of electrochemical redox substrate design, anatomy, benefits, and biosensing potential. A proper understanding of molecular design can lead to the development of a library of novel self-immolative redox molecules that would have huge implications for measurement science and technology.

Inhibited reactivity of horseradish peroxidase by its conjugated proteins through redox mediated electrochemical interrogation

In this article, a classical redox mediated metal (in this study, Au) indicator electrode approach was revisited for the direct interrogation of ‘slow’ enzymatic redox second-order kinetics of horseradish peroxidase (HRP) for oxidative conversion of the model redox substrates, ferrocenemethanol (FcMeOH) and hydroquinone (H2Q) in low HRP-concentration levels. The presented electroanalysis allowed us to recognize the inhibited enzyme reactivity of HRP for FcMeOH-oxidation by its conjugation to model proteins (streptavidin, avidin, and monoclonal antibody), whereas this effect was negligible for H2Q-oxidation by HRP. From a theoretical investigation using finite element analysis, slow kinetics of HRP can be sensitively recognized through an Au indicator electrode approach in the case where HRP bulk electrolyzes a redox substrate in a solution. Five redox substrates were electrochemically investigated, and FcMeOH and H2Q showed negligible side reactions during mediation between HRP and the indicator electrode. From the chronoamperometric approach, the apparent rate constants for oxidative conversion of FcMeOH by the non-conjugated, free HRP were estimated to be ∼104 M−1s−1 at various pH values, while that of H2Q was roughly two orders of magnitude higher at pH ∼7. The molecular docking simulation revealed the need for a significant conformational change of HRP for FcMeOH access to the heme site, whereas the structural alternation of HRP was negligible for access of the H2Q into the domain of the enzyme. The simulation result indicates that the oxidative conversion rate of FcMeOH by HRP could be more susceptible to recognize additionally induced steric hindrance for access of the redox substrate to the catalytic site of the enzyme than that of H2Q. We experimentally verified that the apparent rate constant for FcMeOH-oxidation by the HRP conjugated to streptavidin was estimated to be ∼5 times smaller than that by the free HRP, and those by other conjugates to avidin and monoclonal antibody were ∼60% of that by the non-conjugated one. In contrast to the case of FcMeOH, the estimated reactivity of H2Q by HRP was barely affected by the conjugated model proteins. The presented study demonstrated that a redox enzymatic reactivity of HRP could be significantly affected by conjugated proteins when access of a redox substrate to a catalytic site of an enzyme is susceptible to conformational changes.

Chronic dietary supplementation with nicotinamide riboside reduces sleep need in the laboratory mouse.

Non-rapid eye movement sleep (NREMS) is accompanied by a reduction in cerebral glucose utilization. Enabling this metabolic change may be a central function of sleep. Since the reduction in glucose metabolism is inevitably accompanied by deceleration of downstream oxidation/reduction reactions involving nicotinamide adenine dinucleotide (NAD), we hypothesized a role for NAD in regulating the homeostatic dynamics of sleep at the biochemical level. We applied dietary nicotinamide riboside (NR), a NAD precursor, in a protocol known to improve neurological outcome measures in mice. Long-term (6-10 weeks) dietary supplementation with NR reduced the time that mice spent in NREMS by 17 percent and accelerated the rate of discharge of sleep need according to a mathematical model of sleep homeostasis (Process S). These findings suggest that increasing redox capacity by increasing nicotinamide availability reduces sleep need and increases the cortical capacity for energetically demanding high-frequency oscillations. In turn, this work demonstrates the impact of redox substrates on cortical circuit properties related to fatigue and sleep drive, implicating redox reactions in the homeostatic dynamics of cortical network events across sleep-wake cycles.

Production of bioelectricity may play an important role for the survival of Xanthomonas campestris pv. campestris (Xcc) under anaerobic conditions

The plant pathogen Xanthomonas is commonly found in biocontaminated bioreactors; however, few studies have evaluated the growth and impacts of this microorganism on bioreactors. In this study, we examined the characteristics of Xanthomonas campestris pv. campestris (Xcc). Our results showed that Xcc could reduce metal Fe (III) and decolorise methyl orange in vitro. Moreover, I-t and cyclic voltammetry curves showed that Xcc could generate bioelectricity and had two extracellular electron transfer pathways, similar to that of Shewanella. Based on the spectral analysis of intact cells and scanning electron microscopy analysis, one pathway was speculated to involve cytochrome C by direct contact with the pili or cell surface. The other pathway may involve indirect mediators, such as redox substrates, among extracellular polymeric substances. For the direct extracellular electron transfer process, the charge transfer coefficient α, electron number n, and the electron transfer rate constant ks were determined to be 0.49, 2.6, and 2.2 × 10−3 s−1, respectively. In the indirect extracellular electron transfer processes, the values of α, n, and ks were 0.52, 4, and 1.21 s−1, respectively. Of these two transfer methods, indirect electron transfer is dominant and faster than direct electron transfer. Moreover, after mutation of the dsbD gene, which is important for indirect electron transfer, the electrochemical parameters α, n, and ks decreased. Our findings reveal a new anaerobic mechanism mediating the survival of Xcc during wastewater treatment, and may help develop new strategies for preventing Xcc growth during wastewater treatment.

Extracellular Protein Disulfide Isomerase Cleaves Allosteric Disulfides in Histidine-Rich Glycoprotein to Regulate Thrombus Formation

Introduction The fine-tuning of thrombus formation is influenced by multiple factors among which extracellular protein disulfide isomerase (PDI) released by activated platelets and endothelial cells plays critical roles. However, the precise mechanisms whereby PDI modulates the kinetics of thrombosis remain elusive. Using mechanism-based kinetic trapping strategy, we identified plasma histidine-rich glycoprotein (HRG) as a substrate of extracellular PDI during thrombus formation. HRG exerts both anticoagulant and procoagulant functions. On one hand, HRG inhibits the contact pathway by binding to activated factor XII (fXIIa); on the other hand, HRG attenuates the anticoagulant activity of antithrombin (AT) by competing with AT binding to endothelial heparan sulfate. Both functions are dependent on zinc ions. In this study, we characterized the effects of PDI-mediated disulfide bond cleavage on HRG functions in the context of thrombosis. Methods Recombinant PDI variant with the C-terminal catalytic Cys of the CGHC motif replaced with Ala (PDI-CA) was used to trap its redox substrates in platelet rich plasma (PRP). Dual fluorescent immunoblotting was utilized to detect the stabilized intermediate complex between PDI-CA and HRG. Differential cysteine alkylation and mass spectrometry was performed using purified plasma HRG to identify the disulfide bonds targeted by PDI. ELISA was performed to determine the effects of PDI treatment on HRG binding to heparin, an analog of endothelial heparan sulfate, and fXIIa. Cell-based ELISA, immunofluorescent imaging, and immunohistochemistry were employed to examine in vitro and in vivo binding of HRG and AT on endothelial cells. HRG-mediated inhibition of fXIIa activity was determined using the chromogenic substrate S-2302. The kinetics of HRG accumulation during thrombus formation were examined using high-speed intravital microscopy in the cremasteric arterioles. The effects of HRG on thrombus formation were examined in the laser injury thrombosis model in the presence (wild-type mice) or absence of fXII (f12-/- mice). Results The trapping mutant PDI-CA, but not variants of endoplasmic protein 57 (ERp57), a close member in the PDI family with similar domain structure, formed disulfide-linked complexes with HRG in PRP. Mass spectrometry showed that PDI cleaves three disulfide bonds, C306-C309, C390-C434 and C409-C410, in the histidine-rich region of HRG that is important for its binding to heparan sulfate and fXIIa. Compared to inert-PDI (PDI-AA), where both catalytic Cys were substituted with Ala, wild-type PDI (PDI-CC) increased HRG binding to heparin in a Zn2+-dependent manner. Plasma treated with PDI-CC had increased HRG binding but decreased AT binding to cultured endothelial cells compared to PDI-AA treated control. Further, PDI-CC increased HRG binding to fXIIa and enhanced its inhibitory effect on fXIIa activity. Following laser injury of cremaster arterioles, plasma HRG accumulates rapidly at the injury site preceding the main platelet signal. When mice were treated with Eptifibatide, an integrin αIIbβ3 antagonist that eliminates platelet deposition and Zn2+release, plasma HRG accumulation at the site of vessel injury was reduced, indicating a critical role of Zn2+ for HRG binding in vivo. Intravenous treatment with a PDI inhibitor, isoquercetin, also inhibited HRG accumulation in the growing thrombus. In addition, following FeCl3-induced carotid injury, PDI inhibition by isoquercetin was found to reduce HRG binding but sustain AT binding on the injured artery as determined by immunohistochemistry. Finally, knockdown of plasma HRG with vivo-siRNA in f12-/- mice attenuated thrombus formation compared to scramble siRNA treatment thus suggesting a procoagulant role of HRG independent of fXIIa. Conclusion PDI cleavage of allosteric disulfide bonds in HRG represents a novel regulatory mechanism that fine-tunes the kinetics of thrombus formation. Our results indicate that at the early stage of thrombosis, PDI promotes HRG binding to endothelial cells to suppress the anticoagulant activity of AT and allow the rapid initiation of thrombosis; at the later stage, PDI reduction of HRG enhances its binding to fXIIa leading to inhibition of fXIIa activity to prevent excessive clot formation. Disclosures Bowley: Pfizer: Current Employment.

Nitrate-Dependent Iron Oxidation: A Potential Mars Metabolism.

This work considers the hypothetical viability of microbial nitrate-dependent Fe2+ oxidation (NDFO) for supporting simple life in the context of the early Mars environment. This draws on knowledge built up over several decades of remote and in situ observation, as well as recent discoveries that have shaped current understanding of early Mars. Our current understanding is that certain early martian environments fulfill several of the key requirements for microbes with NDFO metabolism. First, abundant Fe2+ has been identified on Mars and provides evidence of an accessible electron donor; evidence of anoxia suggests that abiotic Fe2+ oxidation by molecular oxygen would not have interfered and competed with microbial iron metabolism in these environments. Second, nitrate, which can be used by some iron oxidizing microorganisms as an electron acceptor, has also been confirmed in modern aeolian and ancient sediment deposits on Mars. In addition to redox substrates, reservoirs of both organic and inorganic carbon are available for biosynthesis, and geochemical evidence suggests that lacustrine systems during the hydrologically active Noachian period (4.1–3.7 Ga) match the circumneutral pH requirements of nitrate-dependent iron-oxidizing microorganisms. As well as potentially acting as a primary producer in early martian lakes and fluvial systems, the light-independent nature of NDFO suggests that such microbes could have persisted in sub-surface aquifers long after the desiccation of the surface, provided that adequate carbon and nitrates sources were prevalent. Traces of NDFO microorganisms may be preserved in the rock record by biomineralization and cellular encrustation in zones of high Fe2+ concentrations. These processes could produce morphological biosignatures, preserve distinctive Fe-isotope variation patterns, and enhance preservation of biological organic compounds. Such biosignatures could be detectable by future missions to Mars with appropriate instrumentation.

The Architecture of Thiol Antioxidant Systems among Invertebrate Parasites.

The use of oxygen as the final electron acceptor in aerobic organisms results in an improvement in the energy metabolism. However, as a byproduct of the aerobic metabolism, reactive oxygen species are produced, leaving to the potential risk of an oxidative stress. To contend with such harmful compounds, living organisms have evolved antioxidant strategies. In this sense, the thiol-dependent antioxidant defense systems play a central role. In all cases, cysteine constitutes the major building block on which such systems are constructed, being present in redox substrates such as glutathione, thioredoxin, and trypanothione, as well as at the catalytic site of a variety of reductases and peroxidases. In some cases, the related selenocysteine was incorporated at selected proteins. In invertebrate parasites, antioxidant systems have evolved in a diversity of both substrates and enzymes, representing a potential area in the design of anti-parasite strategies. The present review focus on the organization of the thiol-based antioxidant systems in invertebrate parasites. Differences between these taxa and its final mammal host is stressed. An understanding of the antioxidant defense mechanisms in this kind of parasites, as well as their interactions with the specific host is crucial in the design of drugs targeting these organisms.

Electrochemical Characterization of some Commercial Screen-Printed Electrodes in Different Redox Substrates

Electrochemical Characterization of some Commercial Screen-Printed Electrodes in Different Redox Substrates

Electrochemical Characterization of some Commercial Screen-Printed Electrodes in Different Redox Substrates

Electrochemical sensors based on screen-printed electrodes (SPEs) are applied for the precise detection of an analyte concentration in a sample solution and is one of the thrust areas of research, especially in healthcare and environmental monitoring. In this article we report electrochemical characterization of some commercially available SPEs obtained from three different commercial manufacturers in three different redox probes such as potassium ferricyanide, ascorbic acid/vitamin-C and nicotinamide adenine dinucleotide. The main aim of this study is to compare the sensitivity, stability, ideality and reproducibility in the development of electrochemical sensors for specific detection of biological or chemical substances using SPEs.

Structure and multistate function of the transmembrane electron transporter CcdA.

The mechanism by which transmembrane reductases use a single pair of cysteine residues to relay electrons between protein substrates across biological membranes is a long-standing mystery in thiol-redox biochemistry. Here we show the NMR structure of a reduced-state mimic of archaeal CcdA, a protein that transfers electrons across the inner membrane, by using a redox-active NMR sample. The two cysteine positions in CcdA are separated by 20 Å. Whereas one is accessible to the cytoplasm, the other resides in the protein core, thus implying that conformational exchange is required for periplasmic accessibility. In vivo mixed disulfide-trapping experiments validated the functional positioning of the cysteines, and in vitro accessibility results confirmed conformational exchange. Our NMR and functional data together show the existence of multiple conformational states and suggest a four-state model for relaying electrons from cytosolic to periplasmic redox substrates.

Sensitive origami dual-analyte electrochemical immunodevice based on polyaniline/Au-paper electrode and multi-labeled 3D graphene sheets

In this work, a sensitive microfluidic origami electrochemical immunodevice (denoted as μ-OECI in this work) has been developed, based on polyaniline-Au nanoparticles (AuNPs) modified paper working electrode (PANI/Au-PWE) as sensor platform and different redox substrates as tracers. Au-PWE was fabricated through the growth of an AuNPs layer on the surfaces of cellulose fibers in the PWE. Through electropolymerization of aniline in an Au-PWE, the effective surface area and the sensitivity of the PANI/Au-PWE were enhanced remarkably. 3D graphene sheets (GS) with high surface-to-volume reaction and good in-plane conductivity were used as matrices to immobilize the redox probes and antibodies. Each biorecognition event yielded a distinct voltammetric peak, whose position and size reflected the identity and level of the corresponding antigen. The large amount of redox probes on the 3D GS greatly amplified the detection signals, and the good biocompatibility of PANI/Au-PWE retained good stability for the sandwich-type immunoassay. Using carcinoembryonic antigen and α-fetoprotein as model analytes, the simultaneous multiplex immunoassay showed linear ranges of over 4 orders of magnitude with the detection limits down to 0.5 and 0.8 pg·mL−1, respectively. This facile biosensing μ-OECI exhibited high sensitivity and specificity, and it was applied in point-of-care testing of other cancer markers for remote regions and developing countries.

A branched electrode based electrochemical platform: towards new label-free and reagentless simultaneous detection of two biomarkers

A branched electrode platform was proposed for label-free and reagentless simultaneous tumor markers detection based on different redox substrates.

Electrochemical deposition of gold at liquid–liquid interfaces studied by thin organic film-modified electrodes

The unique physico-chemical properties of gold nanoparticles portrayed in their chemical stability, the size-dependent electrochemistry, and the unusual optical properties make them suitable modifiers of various surfaces used in the fields of optical devices, electronics, and biosensors. In this work we present two different methods to obtain metallic gold nanoparticles at a liquid–liquid interface, and to control their growth by adjusting the experimental conditions. Decamethylferrocene (DMFC), used as an oxidizable compound dissolved in an organic solvent that is spread as a thin film on the surface of graphite electrode, serves as a redox partner to exchange electrons across the liquid–liquid interface with the other redox counter-partner [AuCl4]− present in the conjoined water phase. The interfacial electron transfer between the DMFC and the [AuCl4]− ions leads to deposition of metallic gold nanoparticles at the liquid–liquid interface. The structure and features of the deposited Au nanoparticles were studied by means of microscopic and voltammetric techniques. The morphology of the Au deposit depends on the concentration ratio of redox partners and both electrode and liquid–liquid interfacial potential differences. Depending on whether the Au deposit was obtained by ex situ (at open circuit potential) or by “in situ” (by cycling of the electrode potential) approach, we observed quite different effects to the ion transfer reactions probed by the thin-film electrode set-up. The possible reasons for the different behavior of the Au nanoparticles are discussed in terms of the structure and the properties of the obtained Au deposit. In separate experiments, we have demonstrated catalytic effects of the Au nanoparticles towards enhancing the electron transfer between DMFC and two aqueous redox substrates, hexacyanoferrate and hydrogen peroxide.

Polyazetidine-Coated Microelectrodes: Electrochemical and Diffusion Characterization of Different Redox Substrates

The present paper reports on the diffusion characteristics and electron transfer properties of a membrane obtained from polyazetidine prepolymer (PAP) consisting of repeating units of 1-(aminomethyl)-1-{2-[(6-oxyhexane)amino]ethyl}-3-hydroxyazetidinium chloride studied in the presence of seven simple redox electroactive molecules: ABTS, catechol, dopamine, ferrocenecarboxylic acid, ferricyanide, ferrocyanide, and the osmium complex bis(2,2-bipyridyl)-4-aminomethylpyridine chloride hexafluorophosphate (Os[(bpy)(2) 4-AMP Cl](+)). Using water as medium, the apparent diffusion coefficients (D(app)), the concentrations of the compounds in the membrane, and the heterogeneous rate constants (k(s)) were calculated as a function of temperature, and the influence thereof on these parameters was evaluated. Even if D(app) and k(s) values in the presence of PAP are smaller than in solution, this decrease is small enough to indicate that the PAP membrane shows excellent diffusion and electron-exchange properties with respect to other commonly used membranes reported in the literature.

Electron-transfer mediation on poly-aryl dendrimer-modified electrodes

Electrochemical properties of a dendrimer-modified electrode that was prepared by immobilization of ferrocenyl-terminated dendrimers on a poly-phenyl acetate anchoring layer were investigated in CH 2Cl 2. The anchoring layer was made by electro-grafting of the corresponding diazonium salt on a glassy carbon surface. The method allows the fabrication of a robust interface where the properties of the dendrimers are well-preserved. Moreover, the control of the layer properties as the permeation of molecules from the solution to the surface could be tuned up from only limited to totally blocked through the electrochemical conditions used during the electro-grafting of the anchoring layer. Detailed investigations performed with cyclic voltammetry and on different types of layers show that the modified electrode catalyses the oxidation of redox substrates. The process depends on the standard potential of the redox couple compared to that of the adsorbed dendrimer molecules. Experiments indicate that the electron exchange with molecules in solution takes place mainly at the dendrimer film–solution interface as the dendrimers inside the film permit the charge-transfer through the modified film to the carbon substrate. The interest of using robust electrode dendrimer relies on the possibility of large structural variations allowing the careful introduction of specific properties in the layer.

Photochemically-, Chemically-, and pH-Controlled Electrochemistry at Functionalized Spiropyran Monolayer Electrodes

A photoisomerizable nitrospiropyran monolayer assembled on a Au electrode provides a functionalized interface for the photochemical, pH, and thermal control of electrochemical processes of charged electroactive redox probes. (Mercaptobutyl)nitrospiropyran 1 was assembled as a monolayer on a Au electrode. The monolayer exhibits reversible photoisomerizable features, and illumination of the nitrospiropyran monolayer, SP state, 320 nm < λ < 350 nm, yields at pH = 7.0 the protonated nitromerocyanine monolayer state, MRH+ state. Further irradiation of the MRH+ monolayer, λ > 495 nm, regenerates the SP state of the monolayer. The light-induced transformation of the monolayer between a neutral and a positively-charged interface allows the control of the electron transfer processes at the electrode interface. Electrooxidation of the negatively-charged (3,4-dihydroxyphenyl)acetic acid, DHPAA, is enhanced at the MRH+ monolayer electrode as compared to the SP-functionalized monolayer electrode. Electrooxidation of the positively-charged 3-hydroxytyramine (dopamine), DOPA, is inhibited at the MRH+ monolayer electrode as compared to its oxidation by the SP monolayer electrode. The control of the electrochemical oxidation of DHPAA and DOPA at the photoisomerizable monolayer electrode is attributed to the electrostatic interactions of the MRH+ monolayer electrode with the redox-active substrates. Electrostatic attraction of DHPAA and repulsion of DOPA by the MRH+ monolayer results in enhancement or inhibition of the electrochemical processes, respectively. By reversible isomerization of the monolayer between the SP and MRH+ states, cyclic amperometric transduction of the optical signals recorded by the monolayer is accomplished. In the presence of a mixture of oppositely-charged redox substrates, i.e. DHPAA and 2,5-bis[[2-(dimethylbutylammonio)ethyl]amino]-1,4-benzoquinone (3) or pyrroloquinoline quinone, PQQ, (4) and 3, photostimulated selective electrochemistry is accomplished in the presence of the photoisomerizable monolayer electrode. The transformation of the protonated nitromerocyanine monolayer, MRH+ state, generated at pH = 7.0, to the zwitterionic nitromerocyanine configuration, MR± state at higher pH, allows the pH-controlled electrooxidation of DHPAA and DOPA at the monolayer electrode. Similarly, thermal isomerization of the SP monolayer electrode, pH = 7.0, 60 °C, yields the MRH+ monolayer electrode. These thermochromic features of the monolayer are employed to respectively activate or deactivate the electrooxidation of DHPAA or DOPA at the functionalized electrode. By cyclic thermal isomerization of the SP monolayer to the MRH+ monolayer followed by photochemical isomerization of the MRH+ monolayer followed by photochemical isomerization of the MRH+ monolayer to the SP state, λ > 495 nm, the thermochromic and photochromic features of the monolayer are amperometrically transduced via the oxidation of DHPAA and DOPA, respectively. Electrochemical oxidation of DHPAA and DOPA is further accomplished by the application of a dinitrospiropyran monolayer (2) electrode in the presence of the dinitrophenyl antibody, DNP-Ab. (Mercaptobutyl)dinitrospiropyran 2 was assembled as a monolayer on a Au electrode. The dinitrospiropyran monolayer, SP state, exhibits antigen features for the DNP-Ab, where the protonated dinitromerocyanine monolayer, MRH+ state, lacks antigen features for the DNP-Ab. Association of the DNP-Ab to the SP monolayer electrode blocks the electrooxidation of DHPAA or DOPA. Photochemical isomerization of the SP monolayer to the MRH+ state, 320 nm < λ < 350 nm, results in the release of DNP-Ab and the activation of the electrooxidation of DHPAA and DOPA. By the reversible photoisomerization of the monolayer between the SP and MRH+ states in the presence of DNP-Ab, cyclic amperometric transduction of the optical signals recorded by the monolayer is accomplished.

Morphology and monoterpene biosynthetic capabilities of secretory cell clusters isolated from glandular trichomes of peppermint (Mentha piperita L.)

Secretory cells were isolated from the monoterpene-producing glandular trichomes (peltate form) of peppermint as clusters of eight cells each. These isolated structures were shown to be non-specifically permeable to low-molecular-weight, water-soluble cofactors and substrates. Short incubation periods with the polar dye Lucifer yellow iodoacetamide (Mr=660) resulted in a uniform staining of the cytoplasm, with exclusion of the dye from the vacuole. The molecular-weight exclusion limit for this permeability was shown to be less than approx. 1800, based on exclusion of fluorescein-conjugated dextran (Mr ∼ 1800). Intact secretory cell clusters very efficiently incorporated [(3)H]geranyl pyrophosphate into monoterpenes. The addition of exogenous cofactors and redox substrates affected the distribution of monoterpenes synthesized from [(3)H]geranyl pyrophosphate, demonstrating that the cell clusters were permeable to these compounds and that the levels of endogenous cofactors and redox substrates were depleted in the isolated cells. When provided with the appropriate cofactors, such as NADPH, NAD(+), ATP, ADP and coenzyme A, the isolated secretory cell clusters incorporated [(14)C]sucrose into monoterpenes, indicating that these structures are capable of the de-novo biosynthesis of monoterpenes from a primary carbon source, and that they maintain a high degree of metabolic competence in spite of their permeable nature.

Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn

The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca(2+) transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive (45)Ca(2+) transport into mitochondria of corn roots. Ca(2+) uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca(2+) uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca(2+) transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca(2+) transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca(2+) uptake. However, ATP-dependent Ca(2+) uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([(14)C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn.