Plant tissue-and photosynthesis-based biosensors.

Biosensors are promising biotools, alternative or complementary to conventional analysis techniques, for fast, simple, cheap and reliable screening. This article reviews the biosensors that use plant components as biorecognition elements. In the first section, plant tissue-based biosensors are summarised and classified according to the enzyme used. Afterwards, photosynthesis-based biosensors, including the types of photosynthetic materials and immobilisation methods, are described.

Nasal nanovaccines.

Nasal administration of vaccines is convenient for the potential stimulation of mucosal and systemic immune protection. Moreover the easy accessibility of the intranasal route renders it optimal for pandemic vaccination. Nanoparticles have been identified as ideal delivery systems and adjuvants for vaccine application. Heterogeneous protocols have been used for animal studies. This complicates the understanding of the formulation influence on the immune response and the comparison of the different nanoparticles approaches developed. Moreover anatomical and immunological differences between rodents and humans provide an additional hurdle in the rational development of nasal nanovaccines. This review will give a comprehensive expertise of the state of the art in nasal nanovaccines in animals and humans focusing on the nanomaterial used.

The effects of drug complexation on the stability and conformation of human serum albumin: protein unfolding.

We report different analytical methods used to study the effects of 3\'-azido-3\'-deoxythymidine, aspirin, taxol, cisplatin, atrazine, 2,4-dichlorophenoxyacetic, biogenic polyamines, chlorophyll, chlorophyllin, poly(ethylene glycol), vanadyl cation, vanadate anion, cobalt-hexamine cation, and As2O3, on the stability and secondary structure of human serum albumin (HSA) in aqueous solution, using capillary electrophoresis, Fourier transform infrared, ultraviolet visible, and circular dichroism (CD) spectroscopic methods. The concentrations of HSA used were 4% to 2% or 0.6 to 0.3 mM, while different ligand concentrations were 1 microM to 1 mM. Structural data showed drugs are mostly located along the polypeptide chains with both specific and nonspecific interactions. The stability of drug-protein complexes were in the order K(VO(2+)) 1.2 x 10(8) M(-1) > K(AZT) 1.9 x 10(6) M(-)1 > K(PEG) 4.1 x 10(5) M(-1) > K(atrazine) 3.5 x 10(4) M(-1) > K(chlorophyll) 2.9 x 10(4) M(-1) > K2,4-D 2.5 x 10(4) M-1 > K(spermine) 1.7 x 10(4) M(-1) > K(taxol) 1.43 x 10(4) M(-1) > K(Co(3+)) > 1.1 x 10(4) M(-1) > K(aspirin) 1.04 x 10(4)i(-1) > K(chlorophyllin) 7.0 x 10(3) M(-1) > K(VO(3)(-)) 6.0 x 103 M(-1) > K(spermidine) 5.4 x 10(3) M(-1) > K(putrescine) 3.9 x 10(3) M(-1) > K(As(2)O(3)) 2.2 x 10(3) M(-1)> K(cisplatin) 1.2 x 10(2) M(-1). The protein conformation was altered (infrared and CD results) with major reduction of alpha-helix from 60 to 55% (free HSA) to 49 to 40% and increase of beta-structure from 22 to 15% (free HSA) to 33 to 23% in the drug-protein complexes. The alterations of protein secondary structure are attributed to a partial unfolding of HSA on drug complexation.

Resveratrol binding to human serum albumin.

Resveratrol (Res), a polyphenolic compound found largely in the skin of red grape and wine, exhibits a wide range of pharmaceutical properties and plays a role in prevention of human cardiovascular diseases [Pendurthi et al., Arterioscler. Thromb. Vasc. Biol. 19, 419-426 (1999)]. It shows a strong affinity towards protein binding and used as inhibitor for cyclooxygenase and ribonuclease reductase. The aim of this study was to examine the interaction of resveratrol with human serum albumin (HSA) in aqueous solution at physiological conditions, using a constant protein concentration (0.3 mM) and various pigment contents (microM to mM). FTIR, UV-Visible, CD, and fluorescence spectroscopic methods were used to determine the resveratrol binding mode, the binding constant and the effects of pigment complexation on protein secondary structure. Structural analysis showed that resveratrol bind non-specifically (H-bonding) via polypeptide polar groups with overall binding constant of K(Res) = 2.56 x 10(5) M(-1). The protein secondary structure, analysed by CD spectroscopy, showed no major alterations at low resveratrol concentrations (0.125 mM), whereas at high pigment content (1 mM), major increase of alpha-helix from 57% (free HSA) to 62% and a decrease of beta-sheet from 10% (free HSA) to 7% occurred in the resveratrol-HSA complexes. The results indicate a partial stabilization of protein secondary structure at high resveratrol content.

Mechanisms allowing protein delivery in nasal mucosa using NPL nanoparticles.

The intranasal administration of proteins using nanoparticles is a promising approach for several applications, especially for mucosal vaccines. Delivery of protein within the epithelial barrier is a key point to elicit an immune response and nano-carrier has to show no toxicity. The aim of this work was to elucidate the interactions of cationic porous nanoparticles loaded with protein delivery for antigen delivery in the nose. We investigated the loading, the cellular delivery and the epithelial transcytosis of proteins associated to these nanoparticles containing an anionic lipid in their core (NPL). NPL were highly endocytosed by airway epithelial cells and significantly improved the protein delivery into the cell. In vitro transcytosis studies showed that NPL did not modify the in vitro epithelial permeability suggesting no toxicity of these carriers. Moreover protein and NPL did not translocate the epithelial barrier. In vivo studies demonstrated that NPL prolonged the nasal residence time of the protein and no NPL were found beyond the epithelial barrier in vivo, precluding a negative side effect. All together these results establish the NPL as a bio-eliminable and optimal vaccine carrier.

Physicochemical characteristics of the terbium-adriamycin complex and its effects on the sinus node automaticity.

The physicochemical characteristics of the terbium-adriamycin complex (terbomycin) were studied. Perturbations in the visible absorption spectrum of adriamycin by terbium (Tb3+) was indicative of formation of the terbomycin complex. The absorption maximum of free adriamycin at 479 nm shifted towards the absorption maximum of terbomycin at 539 nm. The binding of Tb3+ to adriamycin was negligible at acidic pH. At alkaline pH, the affinity of Tb3+ for adriamycin increased. The stoichiometry of binding was estimated to be 0.5; one Tb3+ ion per two adriamycin molecules. Thermodynamic analysis revealed that the spontaneous formation of terbomycin was due to an increase in the entropy of the system. The effects of adriamycin, Tb3+ and terbomycin on sinus node automaticity were studied using sinus node from rats, superfused with modified mammalian Tris-Tyrode's solution (37 degrees C). The sinus node rate was monitored with intracellular microelectrodes. 25 microM Tb3+ increased the sinus node rate. Adriamycin (50 microM) depressed sinus node automaticity. Terbomycin also reduced the sinus node rate. There was no difference between the effects of adriamycin and terbomycin. The chronotropic effect of terbomycin persisted in the presence of atropine.

Interactions of atrazine and 2,4-D with human serum albumin studied by gel and capillary electrophoresis, and FTIR spectroscopy.

The herbicides 6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine (atrazine) and 2,4-dichlorophenoxyacetic acid (2,4-D) are widely used in agricultural practice to fight dicotyledon weeds mainly in maize, cereals, and lucerne. As a result, these compounds are found not only in the plants, soil, and water, but also in the cultivated ground in the following years as well as in agricultural products such as fruits, milk, butter, and sugar beet. The toxicological effects of herbicides occur in vivo, when transported to the target organ through the bloodstream. It has been suggested that human serum albumin (HSA) serves as a carrier protein to transport 2,4-D to molecular targets. This study was designed to examine the interaction of atrazine and 2,4-D with HSA in aqueous solution at physiological pH with herbicide concentrations of 0.0001-1 mM, and final protein concentration of 1% w/v. Gel and capillary electrophoresis, UV-visible and Fourier transform infrared spectroscopic methods were used to determine the drug binding mode, the drug binding constant, and the protein secondary structure in aqueous solution. Structural analysis showed that different types of herbicide-HSA complexes are formed with stoichiometric ratios (drug/protein) of 3:1 and 11:1 for atrazine and 4.5:1 and 10:1 for 2,4-D complexes. Atrazine showed a weak binding affinity (K=3.50 x 10(4) M(-1)), whereas two bindings (K(1)=2.50 x 10(4) M(-1) and K(2)=8.0 x 10(3) M(-1)) were observed for 2,4-D complexes. The herbicide binding results in major protein secondary structural changes from that of the alpha-helix 55% to 45--39% and beta-sheet 22% to 24--32%, beta-anti 12% to 10--22% and turn 11% to 12--15%, in the drug-HSA complexes. The observed spectral changes indicate a partial unfolding of the protein structure, in the presence of herbicides in aqueous solution.

Catechol-mediated effects of harmaline on the action potential of rat atrial fibres.

The mechanism of the transient enhancement of the amplitude of the action potential (AAP) induced by harmaline (HME) was studied in rat atria. The results show that HME increases AAP through an enhancement of the slow component responsible for the last part of the upstroke, which overcomes an inhibitory action on the initial fast component. The stimulatory effect on the slow component is mediated through adrenergic beta receptors and normally masks an alpha-dependent depressant effect.

The action of mercury on the binding of the extrinsic polypeptides associated with the water oxidizing complex of photosystem II.

Mercury (Hg2+), a sulfhydryl group reactant, was used to probe structure-function relationships in photosystem II (PSII). In the present work, we investigated the impact of mercury on the polypeptide composition of PSII submembrane preparations. Electrophoretic analysis revealed that the incubation of the membranes in the presence of mercury produces the depletion of a polypeptide of molecular weight of 33 kDa. This polypeptide corresponds to the extrinsic protein EP33 of the oxygen evolving complex removed following urea treatment. However, the two closely related extrinsic polypeptides of 16 and 23 kDa, usually removed concomitantly after urea treatment, remained unaffected after the mercury treatment. These data demonstrated the existence of an intrinsic binding site for EP23. The molecular mode of action of mercury in the oxygen evolving complex of PSII is discussed.

Demonstration of thermal dissipation of absorbed quanta during energy-dependent quenching of chlorophyll fluorescence in photosynthetic membranes.

When plant leaves or chloroplasts are exposed to illumination that exceeds their photosynthetic capacity, photoprotective mechanisms such as described by the energy-dependent (non-photochemical) quenching of chlorophyll fluorescence are involved. The protective action is attributed to an increased rate constant for thermal dissipation of absorbed quanta. We applied photoacoustic spectroscopy to monitor thermal dissipation in spinach thylakoid membranes together with simultaneous measurement of chlorophyll fluorescence in the presence of inhibitors of opposite action on the formation of delta pH across the thylakoid membrane (tentoxin and nigericin/valinomycin). A linear relationship between the appearance of fluorescence quenching during formation of the delta pH and the reciprocal variation of thermal dissipation was demonstrated. Dicyclohexylcarbodiimide, which is known to prevent protonation of the minor light-harvesting complexes of photosystem II, significantly reduced the formation of fluorescence quenching and the concurrent increase in thermal dissipation. However, the addition of exogenous ascorbate to activate the xanthophyll de-epoxidase increased non-photochemical fluorescence quenching without affecting the measured thermal dissipation. It is concluded that a portion of energy-dependent fluorescence quenching that is independent of de-epoxidase activity can be readily measured by photoacoustic spectroscopy as an increase in thermal deactivation processes.

Mercury inhibition at the donor side of photosystem II is reversed by chloride.

Mercury is an environmental contaminant that strongly inhibits photosynthetic electron transport, photosystem II being the most sensitive target. We investigated in greater detail the effect of mercury using photosystem II submembrane fractions of higher plants. Oxygen evolution was strongly inhibited and variable chlorophyll fluorescence was severely quenched by mercury. Chloride, an inorganic cofactor known to be essential for the optimal function of photosystem II, significantly reversed the inhibitory effect of mercury. However, calcium, another essential cofactor, showed no reversal capacity. It is concluded that on the donor side of PSII, mercury exerts its action by perturbing chloride binding and/or function. Considering the exceptional affinity of mercury for sulfhydryl groups of proteins, the results suggest the implication of cystein residue(s) in maintaining structural and functional integrity of photosystem II.

Secondary structure and thermal stability of the extrinsic 23 kDa protein of photosystem II studied by Fourier transform infrared spectroscopy.

The secondary structure and thermal stability of the extrinsic 23 kDa protein (OEC23) of spinach photosystem II have been characterized in solution between 25 and 75 degrees C using Fourier transform infrared spectroscopy. Quantitative analysis of the amide I band (1700-1600 cm(-1)) shows that OEC23 contains 5% alpha-helix, 37% beta-sheet, 24% turn, and 34% disorder structures at 25 degrees C. No appreciable conformational changes occur below 45 degrees C. At elevated temperatures, the beta-sheet structure is unfolded into the disorder structure with a major conformational transition occurring at 55 degrees C. Implications of these results for the functions of OEC23 in photosystem II are discussed.

A quantitative secondary structure analysis of the 33 kDa extrinsic polypeptide of photosystem II by FTIR spectroscopy.

In chloroplast photosystem II, the extrinsic polypeptide of 33 kDa is involved in the stabilization the Mn cluster in charge of water splitting and in the fulfilment of the Ca(2+)-cofactor requirement for oxygen evolution. The conformational analysis of the purified 33 kDa extrinsic polypeptide was carried out using FTIR spectroscopy with its self-deconvolution and second derivative resolution enhancement as well as curve-fitting procedures. The FTIR spectroscopic results showed that the isolated polypeptide is characterized by a major proportion beta-sheet conformation (36%) with 27% alpha-helix, 24% turn, and 13% beta-antiparallel structures.

The effects of spermine and spermidine on the structure of photosystem II proteins in relation to inhibition of electron transport.

Polyamines (PAs) are ubiquitous in cells of higher plants and play an important role in many biological functions. Because PAs affect photosynthetic oxygen evolution, this study is designed to investigate the interaction of spermine (Spm) and spermidine (Spd) cations with proteins of photosystem II (PSII) using PSII-enriched submembranes fraction with polyamine concentrations of 0.01-10 mM. Fourier transform infrared (FTIR) difference spectroscopy with its self-deconvolution and second derivative resolution enhancement as well as curve-fitting procedures was applied, in order to determine the cation binding mode, the protein conformational changes and the structural properties of cation-protein complexes. It is shown that at low polyamine concentration, cation-protein interaction (H-bonding) is through the polypeptide C=O groups with no major perturbation of the protein secondary structure. As cation concentration increases, the polyamine complexation causes significant alterations of the protein secondary structure with a decrease of the alpha-helical domains from 47% (uncomplexed PSII) up to 37% (cation complexes) and an increase in the beta-sheet structure from 18% (uncomplexed PSII) up to 29% (cation complexes). Correlations between the effects of polyamines on protein secondary structure and on the rate of oxygen evolution in PSII are also established.

Light-induced Fourier transform infrared spectrum of the cation radical P680+.

The structure of the primary electron donor of photosystem II, P680, is still under debate. It is not decided if it is composed of a chlorophyll (Chl) monomer or dimer. In this study, Fourier transform infrared (FTIR) spectroscopy was used to analyze the changes in the vibration modes occurring upon photooxidation of P680 in a Mn-depleted PS II preparation. It is demonstrated that illumination of the above in the presence of artificial electron acceptors results in a light-minus-dark absorbance change typical of the formation of P680+. The light-minus-dark difference FTIR spectrum obtained under similar conditions is characterized by two negative peaks located at 1694 and 1652 or 1626 cm-1 that can be assigned to the 9-keto groups of the P680 Chl, the latter band being indicative of a strongly associated group. These vibrations are shifted to 1714 and 1676 cm-1, respectively, in the positive features of the difference spectrum attributed to P680+. The occurrence of two pairs of bands attributed to 9-keto groups is discussed in terms of P680 being formed of a Chl dimer.

Effects of harmine on transmembrane potentials of guinea-pig atrial muscle.

1 The effects of harmine 8.3 X 10(-5) M on membrane potentials of guinea-pig atrial muscle were analyzed and compared with those of harmaline. Transmembrane potentials of contractile fibres were measured during exposure to the drug at 30 degrees C. 2 In preparations superfused with 5.4 mmol K+-Tyrode, harmine produced a progressive reduction in the amplitude of the action potential (AP), in the absence of any change in resting potential (RP) and a prolongation of the duration of the action potential (APD). 3 Harmaline produced an enhancement of AP; RP was not affected and APD was prolonged. 4 The amplitude of slow responses elicited by noradrenaline in 16.2 mmol K+-Tyrode was enhanced by harmine. 5 It is proposed that dehydrogenation of harmaline to harmine reverses the initial stimulatory action of harmaline on AP because the depressant action on the fast component of the upstroke prevails over the stimulatory effect on the slow component.

The effect of harmine on the action potential of the guinea-pig atrial muscle depends on the external calcium concentration.

1 The influence of the external calcium concentration on the effect of harmine 2 x 10(-5) M upon the guinea-pig atrial muscle was analysed. Transmembrane potentials of contractile fibres were measured during exposure to the drug at 30 degrees C. 2 In preparations superfused with 1.35 mM Ca2+-Tyrode solution and driven at 60/min (1 Hz) harmine depressed the amplitude of the action potential (AP) and the maximum velocity of the upstroke (dV/dt). The resting potential was not affected. Harmine depressed similarly the dV/dt of fibres superfused with 2.7 mM Ca2+-Tyrode solution but the AP was slightly enhanced. 3 Harmine diminished both the AP and the dV/dt of fibres superfused with 2.7 mM Ca2+-Tyrode solution and driven at a fast rate (180/min, 3 Hz). Increased external calcium concentration (5.4 mM) annulled the depressant effect on Ap while the action on dV/dt persisted. 4 It is concluded that the effect of harmine on the Ap depends on the external calcium concentration. Increase [Ca2+]o reverses the depressant effect of harmine because it annuls the effect of the drug on the slow component of the upstroke. The action on the initial fast component of the rising phase of the action potential persists.

Effects of harmala alkaloids on transmembrane potentials of guinea-pig papillary muscles.

1 The significance of the presence of a methoxy group in the 7 position of the indole nucleus of harmala alkaloids in terms of their effects on the action potential of cardiac muscle was analysed. Guinea-pig papillary muscles were superfused with Tyrode solution at 30 degrees C and exposed to harmine or its analogue harmane, in which the methoxy group has been removed from the molecule. 2 Harmine 2 x 10(-5) M enhanced the amplitude of the action potential (AAP) of normal fibres and of slow responses elicited by noradrenaline in fibres depolarized by 21.6 mM K+-Tyrode. The effect of harmine on AAP occurred in the absence of any change in membrane resting potential or maximum velocity of the upstroke and it was abolished by propranolol. Harmane 2 x 10(-5) M did not have any effect on normal action potentials and slow responses. 3 Higher concentrations of the two analogues depressed both AAP and the maximum velocity of the upstroke of the action potential, without affecting the duration of the action potential. 4 It is concluded that: (a) removal of the methoxy group from harmine (1) abolishes the catechol-mediated stimulatory effect of a low concentration of the drug on the slow component of the upstroke of the action potential, and (2) does not modify the depressant effect of a high concentration of the drug. (b) The two analogues, harmine and harmane, do not affect the duration of the action potential of ventricular muscle.

Heterogeneity of Photosystem I reaction centers in barley leaves as related to the donation from stromal reductants.

The light-response curves of P700 oxidation and time-resolved kinetics of P700(+) dark re-reduction were studied in barley leaves using absorbance changes at 820 nm. Leaves were exposed to 45 degrees C and treated with either diuron or diuron plus methyl viologen (MV) to prevent linear electron flow from PS II to PS I and ferredoxin-dependent cyclic electron flow around PS I. Under those conditions, P700(+) could accept electrons solely from soluble stromal reductants. P700 was oxidized under weak far-red light in leaves treated with diuron plus MV, while identical illumination was nearly ineffective in diuron-treated leaves in the absence of MV. When heat-exposed leaves were briefly illuminated with strong far-red light, which completely oxidized P700, the kinetics of P700(+) dark reduction was fitted by a single exponential term with half-time of about 40 ms. However, two first-order kinetic components of electron flow to P700(+) (fast and slow) were found after prolonged leaf irradiation. The light-induced modulation of the kinetics of P700(+) dark reduction was reversed following dark adaptation. The fast component (half time of 80-90 ms) was 1.5 larger than the slow one (half time of about 1 s). No kinetic competition occurred between two pathways of electron donation to P700(+) from stromal reductants. This suggests the presence of two different populations of PS I.

Frequency dependence of the effect of harmine on the duration of the action potential of guinea pig atrial muscle.

The effect of harmine on the duration of the action potential (APD) of guinea pig atrial muscle is analyzed. Harmine prolongs APD through both a depressant effect on automaticity and a direct effect on atrial repolarization. In atria driven at a constant rate: (a) the direct effect becomes less with a faster drive; (b) an increase in concentration of harmine does not result in a greater prolongation of APD. Increased extracellular Ca2+ concentration annulles the APD-prolonging effect.