FAD Group Research Articles

Female rats consuming an iron and omega-3 fatty acid deficient diet preconception require combined iron and omega-3 fatty acid supplementation for the prevention of bone impairments in offspring.

We previously showed in rats that pre- and postnatal deficiencies in iron and omega-3 (n-3) fatty acids can impair bone development, with additive and potentially irreversible effects when combined. This study aimed to investigate, in female rats consuming a combined iron and n-3 fatty acid deficient (ID + n-3 FAD) diet preconception, whether supplementation with iron and docosahexaenoic/eicosapentaenoic acid (DHA/EPA), alone and in combination, can prevent bone impairments in offspring. Using a 2 × 2 factorial design, female Wistar rats consuming an ID + n-3 FAD diet preconception were randomised to receive an: 1) iron supplemented (Fe + n-3 FAD), 2) DHA/EPA supplemented (ID + DHA/EPA), 3) Fe + DHA/EPA, or 4) ID + n-3 FAD diet from gestational day 10 throughout pregnancy and lactation. Post-weaning, offspring (n = 24/group; male:female = 1:1) remained on the respective experimental diets for three weeks until postnatal day 42-45. Offspring born to female rats consuming a control diet preconception and an Fe+DHA/EPA diet throughout pregnancy and lactation served as non-deficient reference group (Control+Fe+DHA/EPA). Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry and bone strength using three-point bending tests. Only offspring in the Fe+DHA/EPA group had significantly higher spine and femur BMD, and higher femur stiffness than offspring in the ID + n-3 FAD group, and had similar spine BMD and femur stiffness as the Control + Fe + DHA/EPA group. Offspring in the Fe + DHA/EPA group further had significantly higher femur strength (ultimate load) than the other experimental groups, and a similar femur strength as the Control + Fe + DHA/EPA group. This study shows that only combined iron and DHA/EPA supplementation can prevent bone impairments in offspring of female rats consuming an iron and n-3 FA deficient diet preconception.

Fad2Vec: 판매 데이터 임베딩 기반 패션 산업 단발성 유행 탐지

In the fashion industry, fads, a phenomenon in which demand for specific items surges in a short time and then disappears, are often observed. However, the conventional demand forecasting methods have limitations in predicting fads because it focuses on long-term trends. This paper presents a new approach called Fad2Vec, which adapts the Item2Vec approach to detect fads effectively. Hot periods composed of a hot point and a preceding period for each product are identified based on time-series sales data. Products are embedded into vectors representing the time points when they are temporarily fashionable. Then, they are clustered into a fad group where fashion items belong to the same cluster and show similar fad patterns. A case study of four famous fashion brands is provided to show how Fad2Vec works and verify its validity. The proposed Fad2Vec approach is expected to be practically utilized for the production planning of fast fashion companies.

Electrophysiological and Imaging Calcium Biomarkers of Aging in Male and Female 5×FAD Mice.

Background:In animal models and tissue preparations, calcium dyshomeostasis is a biomarker of aging and Alzheimer’s disease that is associated with synaptic dysfunction, neuritic pruning, and dysregulated cellular processes. It is unclear, however, whether the onset of calcium dysregulation precedes, is concurrent with, or is the product of pathological cellular events (e.g., oxidation, amyloid-β production, and neuroinflammation). Further, neuronal calcium dysregulation is not always present in animal models of amyloidogenesis, questioning its reliability as a disease biomarker.Objective:Here, we directly tested for the presence of calcium dysregulation in dorsal hippocampal neurons in male and female 5×FAD mice on a C57BL/6 genetic background using sharp electrodes coupled with Oregon-green Bapta-1 imaging. We focused on three ages that coincide with the course of amyloid deposition: 1.5, 4, and 10 months old.Methods:Outcome variables included measures of the afterhyperpolarization, short-term synaptic plasticity, and calcium kinetics during synaptic activation. Quantitative analyses of spatial learning and memory were also conducted using the Morris water maze. Main effects of sex, age, and genotype were identified on measures of electrophysiology and calcium imaging.Results:Measures of resting Oregon-green Bapta-1 fluorescence showed significant reductions in the 5×FAD group compared to controls. Deficits in spatial memory, along with increases in Aβ load, were detectable at older ages, allowing us to test for temporal associations with the onset of calcium dysregulation.Conclusion:Our results provide evidence that reduced, rather than elevated, neuronal calcium is identified in this 5×FAD model and suggests that this surprising result may be a novel biomarker of AD.

Topography of human cytochrome b5/cytochrome b5 reductase interacting domain and redox alterations upon complex formation

Cytochrome b5 is the main electron acceptor of cytochrome b5 reductase. The interacting domain between both human proteins has been unidentified up to date and very little is known about its redox properties modulation upon complex formation. In this article, we characterized the protein/protein interacting interface by solution NMR and molecular docking. In addition, upon complex formation, we measured an increase of cytochrome b5 reductase flavin autofluorescence that was dependent upon the presence of cytochrome b5. Data analysis of these results allowed us to calculate a dissociation constant value between proteins of 0.5±0.1μM and a 1:1 stoichiometry for the complex formation. In addition, a 30mV negative shift of cytochrome b5 reductase redox potential in presence of cytochrome b5 was also measured. These experiments suggest that the FAD group of cytochrome b5 reductase increase its solvent exposition upon complex formation promoting an efficient electron transfer between the proteins.

Preclinical identification of vaccine induced protective correlates in human leukocyte antigen expressing transgenic mice infected with Coccidioides posadasii

There is an emerging interest to develop human vaccines against medically-important fungal pathogens and a need for a preclinical animal model to assess vaccine efficacies and protective correlates. HLA-DR4 (DRB1∗0401 allele) transgenic mice express a human major histocompatibility complex class II (MHC II) receptor in such a way that CD4+ T-cell response is solely restricted by this human molecule. In this study HLA-DR4 transgenic mice were immunized with a live-attenuated vaccine (ΔT) and challenged by the intranasal route with 50–70 Coccidioides posadasii spores, a potentially lethal dose. The same vaccination regimen offers 100% survival for C57BL/6 mice. Conversely, ΔT-vaccinated HLA-DR4 mice displayed 3 distinct manifestations of Coccidioides infection including 40% fatal acute (FAD), 30% disseminated (DD) and 30% pulmonary disease (PD). The latter 2 groups of mice had reduced loss of body weight and survived to at least 50days postchallenge (dpc). These results suggest that ΔT vaccinated HLA-DR4 mice activated heterogeneous immunity against pulmonary Coccidioides infection. Vaccinated HLA-DR4 mice displayed early expansion of Th1 and Th17 cells and recruitment of inflammatory innate cells into Coccidioides-infected lungs during the first 9dpc. While contraction rates of Th cells and the inflammatory response during 14–35dpc significantly differed among the 3 groups of vaccinated HLA-DR4 mice. The FAD group displayed a sharply reduced Th1 and Th17 response, while overwhelmingly recruiting neutrophils into lungs during 9–14days. The FAD group approached moribund by 14dpc. In contrast, vaccinated HLA-DR4 survivors gradually contracted Th cells and inflammatory response with the greatest rate in the PD group. While vaccinated HLA-DR4 mice are susceptible to Coccidioides infection, they are useful for evaluation of vaccine efficacy and identification of immunological correlates against this mycosis.

Complexity and synchronicity of resting state blood oxygenation level-dependent (BOLD) functional MRI in normal aging and cognitive decline

To explore the use of approximate entropy (ApEn) as an index of the complexity and the synchronicity of resting state blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) in normal aging and cognitive decline associated with familial Alzheimer's disease (fAD).Resting state BOLD fMRI data were acquired at 3T from two independent cohorts of subjects consisting of healthy young (age 23 ± 2 years, n = 8) and aged volunteers (age 66 ± 3 years, n = 8), as well as 22 fAD associated subjects (14 mutation carriers, age 41.2 ± 15.8 years; and eight nonmutation carrying family members, age 28.8 ± 5.9 years). Mean ApEn values were compared between the two age groups and correlated with cognitive performance in the fAD group. Cross-ApEn (C-ApEn) was further calculated to assess the asynchrony between precuneus and the rest of the brain.Complexity of brain activity measured by mean ApEn in gray and white matter decreased with normal aging. In the fAD group, cognitive impairment was associated with decreased mean ApEn in gray matter as well as decreased regional ApEn in right precuneus, right lateral parietal regions, left precentral gyrus, and right paracentral gyrus. A pattern of asynchrony between BOLD fMRI series emerged from C-ApEn analysis, with significant regional anti-correlation with cross-correlation coefficient of functional connectivity analysis.ApEn and C-ApEn may be useful for assessing the complexity and synchronicity of brain activity in normal aging and cognitive decline associated with neurodegenerative diseases.

Comparison of Four Different Analgesic Discogram Protocols Comparing the Incidence of Reported Pain Relief Following Local Anesthetic Injection into Concordantly Painful Lumbar Intervertebral Discs

To compare the incidence of pain relief following injection of local anesthetic (LA) into lumbar discs that caused concordant pain during provocation testing. Prospective collected data review from two centers and compare with published results. We compared subjective reported pain relief following provocative testing using the following protocols at three separate facilities: 23 patients undergoing routine provocative discography using contrast alone (PD); 47 patients undergoing provocative discography performed using an equal combination of LA and contrast (CPD); 120 patients injected with LA following routine PD (ADPD); 33 patients undergoing stand-alone analgesic discography (SAAD); and 28 patients injected with LA through a catheter (FAD) placed during provocative discogaphy testing. Pressure-controlled PD showed a positive response rate of 34% per disc in patients with a clinical diagnosis of discogenic pain. None of the PD group without LA had pain relief and less than 10% of the CPD group reported pain relief. Forty percent of the SAAD group with positive pain reproduction reported ≥50% relief and 20% reporting ≥80% relief. Forty-six percent of the ADPD group reported ≥50% relief and 30% reporting ≥80% relief. The FAD group had a greater 80% patients reporting ≥50% pain relief although fewer 26% reporting more convincing ≥80% relief. If the criterion standard to confirm painful annular tears is concordant pain provocation and 80% or greater pain relief following LA injected into lumbar discs, the SAAD, ADPD, and FAD protocols show statistically similar 20% to 30% prevelance.

Influence of PAS Domain Flanking Regions on Oligomerisation and Redox Signalling By NifL

Per-ARNT-Sim (PAS) domains constitute a typically dimeric, conserved α/β tertiary fold of approximately 110 amino acids that perform signalling roles in diverse proteins from all kingdoms of life. The amino terminal PAS1 domain of NifL from Azotobacter vinelandii accommodates a redox-active FAD group; elevation of cytosolic oxygen concentrations result in FAD oxidation and a concomitant conformational re-arrangement that is relayed via a short downstream linker to a second PAS domain, PAS2. At PAS2, the signal is amplified and passed on to effector domains generating the ‘on’ (inhibitory) state of the protein. Although the crystal structure of oxidised PAS1 reveals regions that contribute to the dimerisation interface, 21 amino acids at the extreme N-terminus of NifL, are unresolved. Furthermore, the structure and function of the linker between the two PAS domains has not been determined. In this study we have investigated the importance to signalling of residues extending beyond the core PAS fold. Our results implicate the N-terminus of PAS1 and the helical linker connecting the two PAS domains in redox signal transduction and demonstrate a role for these flanking regions in controlling the oligomerisation state of PAS1 in solution.

Folate-deficiency induced cell-specific changes in the distribution of lymphocytes and granulocytes in rats

Folate (vitamin B(9)) plays key roles in cell growth and proliferation through regulating the synthesis and stabilization of DNA and RNA, and its deficiency leads to lymphocytopenia and granulocytopenia. However, precisely how folate deficiency affects the distribution of a variety of white blood cell subsets, including the minor population of basophils, and the cell specificity of the effects remain unclear. Therefore, we examined the effects of a folate-deficient diet on the circulating number of lymphocyte subsets [T-lymphocytes, B-lymphocytes, and natural killer (NK) cells] and granulocyte subsets (neutrophils, eosinophils, and basophils) in rats. Rats were divided into two groups, with one receiving the folate-deficient diet (FAD group) and the other a control diet (CON group). All rats were pair-fed for 8 weeks. Plasma folate level was dramatically lower in the FAD group than in the CON group, and the level of homocysteine in the plasma, a predictor of folate deficiency was significantly higher in the FAD group than in the CON group. The number of T-lymphocytes, B-lymphocytes, and NK cells was significantly lower in the FAD group than in the CON group by 0.73-, 0.49-, and 0.70-fold, respectively, indicating that B-lymphocytes are more sensitive to folate deficiency than the other lymphocyte subsets. As expected, the number of neutrophils and eosinophils was significantly lower in the FAD group than in the CON group. However, the number of basophils, the least common type of granulocyte, showed transiently an increasing tendency in the FAD group as compared with the CON group. These results suggest that folate deficiency induces lymphocytopenia and granulocytopenia in a cell-specific manner.

Effects of guanidinium ions on the conformational structure of glucose oxidase studied by electrochemistry, spectroscopy, and theoretical calculations: towards developing a chemical-induced protein conformation assay

Understanding conformation transitions of proteins in the presence of a chemical denaturant is a topic of great interest because the rich information contained in chemical unfolding is of fundamental importance for proteomic and pharmaceutical research. In this work, the conformational structure changes of glucose oxidase (GOx) induced by guanidinium ions (Gdm(+)) were studied in detail by a combination of electrochemical methods, various spectroscopic techniques including ultraviolet-visible (UV-vis) absorption, fluorescence, Fourier transform infrared (FTIR), and circular dichroism (CD) spectroscopy, molecular dynamics (MD) simulations, and density functional theory (DFT) calculations with the purpose of revealing the mechanism of chemical unfolding of proteins. The results indicated that GOx underwent substantial conformational changes both at the secondary and tertiary structure levels after interacting with Gdm(+) ions. The interaction of GOx with the chemical denaturant resulted in a disturbance of the structure of the flavin prosthetic group (FAD moiety) that induced the moiety to become less exposed to solvent than that in the native protein molecule. The calculation from quantitative second-derivative infrared and CD spectra showed that Gdm(+) ions induced the conversion of α-helix to β-sheet structures. MD simulations and DFT calculations revealed that Gdm(+) ions could enter the active pocket of the GOx molecule and interact with the FAD group, leading to a significant alteration in the structural characteristics and hydrogen bond networks formed between FAD and the surrounding amino acid residues. These alterations in the conformational structure of GOx resulted in a significant decrease in the catalytic activity of the enzyme to glucose oxidation. The study essentially provides an effective way for investigating the mechanism of chemical denaturant-induced protein unfolding, and this approach can be used for assessing the effect of drug molecules on proteins.

Substitutions in the redox‐sensing PAS domain of the NifL regulatory protein define an inter‐subunit pathway for redox signal transmission

The Per-ARNT-Sim (PAS) domain is a conserved α/β fold present within a plethora of signalling proteins from all kingdoms of life. PAS domains are often dimeric and act as versatile sensory and interaction modules to propagate environmental signals to effector domains. The NifL regulatory protein from Azotobacter vinelandii senses the oxygen status of the cell via an FAD cofactor accommodated within the first of two amino-terminal tandem PAS domains, termed PAS1 and PAS2. The redox signal perceived at PAS1 is relayed to PAS2 resulting in conformational reorganization of NifL and consequent inhibition of NifA activity. We have identified mutations in the cofactor-binding cavity of PAS1 that prevent 'release' of the inhibitory signal upon oxidation of FAD. Substitutions of conserved β-sheet residues on the distal surface of the FAD-binding cavity trap PAS1 in the inhibitory signalling state, irrespective of the redox state of the FAD group. In contrast, substitutions within the flanking A'α-helix that comprises part of the dimerization interface of PAS1 prevent transmission of the inhibitory signal. Taken together, these results suggest an inter-subunit pathway for redox signal transmission from PAS1 that propagates from core to the surface in a conformation-dependent manner requiring a flexible dimer interface.

Colloidal laponite nanoparticles: Extended application in direct electrochemistry of glucose oxidase and reagentless glucose biosensing

The direct electron transfer (DET) between glucose oxidase (GOD) and the underlying glassy carbon electrode (GCE) can be readily achieved via colloidal laponite nanoparticles as immobilization matrix. Cyclic voltammetry of laponite/GOD/GCE, in anaerobic phosphate buffer solution (PBS, 0.1 M, pH 5.0), showed a pair of stable and quasi-reversible peaks at potentials E pa = −0.372 V and E pc = −0.391 V vs. SCE, provoked by the prosthetic FAD group linked to the protein. The electrochemical reaction of laponite/GOD/GCE exhibited a surface-controlled process with the apparent heterogeneous electron transfer rate constant ( k s) of 6.52 s −1 and charge-transfer coefficient ( α) of 0.5. The experiments of FTIR and UV–vis spectroscopy demonstrate that the immobilized GOD on colloidal laponite nanoparticles retained its native structure and its biocatalytic ability to its substrates. Based on the decrease of oxygen electrocatalytic signal, the proposed laponite/GOD/GCE was successfully applied in the reagentless glucose sensing at −0.45 V. The proposed electrode exhibited fast amperometric response (8 s), broad linear range (2.0 × 10 −5–1.9 × 10 −3 M), good sensitivity (4.8 ± 0.5 mA M −1 cm −2), low detection limit (1.0 × 10 −5 M) at a signal-to-noise ratio of 3, and excellent selectivity.

Using BioTek's Synergy™ HT Reader to Measure Reactive Oxygen Species (ROS) Generation in Stimulated Cells

The investigation of biological agents that can stimulate or inhibit cell growth is of great interest to cellular biologists. One such measure is the formation of Reactive Oxygen Species (ROS) as a result of cell stimulation. While there are numerous methodologies to assess ROS species, the use of fluorescent probes has become the preferred method. Here we describe the measurement of DCF fluorescence using a SynergyTM HT to detect and analyze the production of ROS as a result of cell growth stimulation.

A role for cytochrome c and cytochrome c peroxidase in electron shuttling from Erv1

Erv1 is a flavin-dependent sulfhydryl oxidase in the mitochondrial intermembrane space (IMS) that functions in the import of cysteine-rich proteins. Redox titrations of recombinant Erv1 showed that it contains three distinct couples with midpoint potentials of -320, -215, and -150 mV. Like all redox-active enzymes, Erv1 requires one or more electron acceptors. We have generated strains with erv1 conditional alleles and employed biochemical and genetic strategies to facilitate identifying redox pathways involving Erv1. Here, we report that Erv1 forms a 1:1 complex with cytochrome c and a reduced Erv1 can transfer electrons directly to the ferric form of the cytochrome. Erv1 also utilized molecular oxygen as an electron acceptor to generate hydrogen peroxide, which is subsequently reduced to water by cytochrome c peroxidase (Ccp1). Oxidized Ccp1 was in turn reduced by the Erv1-reduced cytochrome c. By coupling these pathways, cytochrome c and Ccp1 function efficiently as Erv1-dependent electron acceptors. Thus, we propose that Erv1 utilizes diverse pathways for electron shuttling in the IMS.

Direct electrochemistry of hemoglobin and glucose oxidase in electrodeposited sol–gel silica thin films on glassy carbon

This work points out that electrogeneration of silica gel (SG) films on glassy carbon electrodes (GCEs) can be applied to immobilize biomolecules – hemoglobin (Hb) or glucose oxidase (GOD) or both of them in mixture – without preventing their activity. These proteins were physically entrapped in the sol–gel material in the course of the electro-assisted deposition process applied to form the thin films onto the electrode surface. SG films were prepared from a precursor solution by applying a suitable cathodic potential likely to induce a local pH increase at the electrode/solution interface, accelerating thereby polycondensation of the silica precursors with concomitant film formation. Successful immobilization of proteins was checked by various physico-chemical techniques. Both Hb and GOD were found to undergo direct electron transfer, as demonstrated by cyclic voltammetry. GCE–SG–Hb gave rise to well-defined peaks at potentials E c = −0.29 V and E a = −0.17 V in acetate buffer, corresponding to the Fe III/Fe II redox system of heme group of the protein, while GCE–SG–GOD was characterized by the typical signals of FAD group at E c = −0.41 V and E a = −0.33 V in phosphate buffer. These two redox processes were also evidenced on a single voltammogram when both Hb and GOD were present together in the same SG film. Hb entrapped in the silica thin film displayed an electrocatalytic behavior towards O 2 and H 2O 2 in solution, respectively in the mM and μM concentration ranges. Immobilized GOD kept its biocatalytic properties towards glucose. Combined use of these two proteins in mixture has proven to be promising for detection of glucose in solution via the electrochemical monitoring of oxygen consumption (decrease of the oxygen electrocatalytic signal).

Effect of a Fish Oil and Arginine-Fortified Diet in Thermally Injured Patients

Burn injury induces a hypercatabolic inflammatory state, predisposing burn patients to malnutrition, poor wound healing, and infectious complications. We conducted this study to determine what effect a diet fortified with fish oil and arginine (FAD) would have on wound healing in a thermally injured population. Twenty-three thermally injured patients were enrolled in this randomized double blind enteral feeding study from July 2002 to August 2004. All study patients received isonitrogenous enteral intragastric feeding within 48 hours of admission. Patients were randomized to our standard diet (STD, ProBalance with Promix, Probalance from Nestlé, Glendale, CA; ProMix R.D., Navaco Laboratories, Phoenix, AZ) or a diet fortified with fish oil and arginine (FAD, Crucial, Nestlé Nutrition Glendale, CA) Diets were advanced as tolerated to meet 100% of estimated needs. The primary endpoint of the study was time to heal the first donor site. There were no statistical differences between the study groups with respect to baseline characteristics. Both diets were well tolerated, and there were no differences in the daily total kilocalories or protein intake per kilogram between the two diet groups throughout the study. Although nonsignificant, the patients in the FAD group showed a slightly faster healing time than those in the STD group (10.8 +/- 2.7 days vs 12.3 +/- 5.2 days, respectively). This trend was further accelerated when those with body surface area burns less than 30% were examined (patients with body surface area burns <30% in the FAD healed in 9.0 +/- 1.7 vs corresponding patients in the standard group who healed in 12.2 +/- 6.2, P = .63). Patients in the FAD group trended to more infections and more adverse complications. The adverse complications were predominantly associated with inhalation injuries. The role of fortified enteral diets in the outcomes of thermally injured patients deserves further study. Such a future study should be conducted in a multicenter trial and involve inhalation injury stratification systems to accurately score and randomize patients for inhalation injury. Finally, the frequency and pattern of infections in patients receiving fortified enteral diets deserves further evaluation.

Study of the Spin−Spin Interactions between the Metal Centers ofDesulfovibrio gigasAldehyde Oxidoreductase: Identification of the Reducible Sites of the [2Fe-2S]1+,2+Clusters

The aldehyde oxidoreductase from Desulfovibrio gigas belongs to the family of molybdenum hydroxylases. Besides a molybdenum cofactor which constitutes their active site, these enzymes contain two [2Fe-2S](2+,1+) clusters which are believed to transfer the electrons provided by the substrate to an acceptor which is either a FAD group or an electron-transferring protein. When the three metal centers of D. gigas AOR are simultaneously paramagnetic, splittings due to intercenter spin-spin interactions are visible when the EPR spectra are recorded at low temperatures. By studying quantitatively these interactions with a model based on the X-ray crystal structure, which takes into consideration the interactions between the magnetic moments carried by all the metal sites of the system, it is possible to determine the location of the reducible sites of the [2Fe-2S] clusters. When combined with the electron-transfer pathways proposed on the basis of the X-ray crystal structure, the results provide a detailed description of the electron-transfer system of D. gigas AOR.

Electron Transfer Reactions of Glucose Oxidase at Au(111) Electrodes Modified with Phenothiazine Derivatives

The catalytic reaction of glucose oxidase (GOx) mediated by 3-(10-phenothiazyl)propionic acid (PT-PA) and phenothiazine-labeled poly(ethylene oxide) (PT-PEO1000) that are covalently bonded to Au(111) electrodes has been investigated. The PT-PA and PT-PEO1000 are reacted with 2-aminoethanethiol (AET), followed by the formation of a self-assembled monolayer (SAM) onto the Au surface. The PT group immobilized on the SAM of AET acts as an effective mediator for the electron transfer (ET) between the electrode and the FAD center of freely diffusing GOx in solution. The ET rate constant estimated from the catalytic current using a newly derived equation is larger by 1 order of magnitude for the PT-PA-modified system (1.1 x 10(5) dm(3) mol(-1) s(-1)) than for the PT-PEO1000 system (1.4 x 10(4) dm(3) mol(-1) s(-1)). The order of the magnitude of the ET rate constant clearly contrasts with the GOx hybrid systems that we previously investigated (Anal. Chem. 2003, 75, 910-917), in which the presence of the PEO spacer enhances the ET reaction rate. The reduction in the apparent PT concentration at the electrode interface due to the high mobility of the PEO chain, leading to low efficiency in the formation of an enzyme-mediator complex, is a possible reason for the lower mediation ability of PT-PEO1000 than that of PT-PA for the ET between the FAD group and PT(+) immobilized on the electrode. Inhibition of the penetration of GOx molecules into the monolayer and of the accessibility of some part of PT groups to GOx molecules could also be reasons for the lower mediation ability of PT-PEO1000 thickly modified on the electrode.

Role of hydrophobic interactions in the flavodoxin mediated electron transfer from photosystem I to ferredoxin-NADP+ reductase in Anabaena PCC 7119.

Hydrophobic interactions play an active role in effective complex formation between ferredoxin-NADP(+) reductase (FNR) and ferredoxin (Fd) from Anabaena, where an aromatic amino acid residue on the Fd surface (F65) and three hydrophobic residues (L76, L78, and V136) on the reductase surface have been shown to be essential for the efficient electron transfer (ET) reaction between Fd and FNR (Martínez-Júlvez et al. (2001) J. Biol. Chem. 276, 27498-27510). Since in this system flavodoxin (Fld) can efficiently replace Fd in the overall ET process, we have further investigated if such hydrophobic interactions are also critical in complex stabilization and ET in the FNR/Fld association. Different ET behaviors with Fld are observed for some of the mutations made at L76, L78, and V136 of Anabaena FNR. Thus, the ET interaction with Fld is almost completely lost upon introduction of negatively charged side chains at these positions, while more conservative changes in the hydrophobic patch can influence the rates of ET to and from Fld by altering the binding constants and the midpoint redox potentials of the flavin group. Therefore, our results confirm that nonpolar residues in the region close to the FAD group in FNR participate in the establishment of interactions with Fld, which serve to orient the two flavin groups in a manner such that ET is favored. In an attempt to look for the counterpart region of the Fld surface, the effect produced by the replacement of the only two nonpolar residues on the Fld surface, I59 and I92, by a Lys has also been analyzed. The results obtained suggest that these two hydrophobic residues are not critical in the interaction and ET processes with FNR. The reactivity of these I92 and I59 Fld mutants toward the membrane-anchored photosystem I (PSI) complex was also analyzed by laser flash absorption spectroscopy. From these data, significant effects are evident, especially for the I92 position of Fld, both in the association constant for complex formation and in the electron-transfer rate constant in the PSI/Fld system.

Direct electron transfer involving a large protein: glucose oxidase

Electrochemical behavior of glucose oxidase fixed in the bulk of a graphite paste electrode, with no additives available to play the role of mediator or redox activator, shows the oxido reduction of the prosthetic FAD group that is still attached to the apoenzyme with no loss of enzyme activity.