In-sights into the effect of heavy metal stress on the endogenous mustard cystatin.

Phytocystatins have been ascribed several protective roles against abiotic and biotic stress conditions. It was, therefore, thought worthwhile to document the effect of heavy metal stress on the endogenous plant cystatin. The mustard cystatin, purified from Brassica juncea (B. juncea) seeds retained its functional property of cysteine proteinase inhibition, despite exposure to high concentrations of metal ions, Cd2+ and Ni2+. An increase in inhibitory activity, ∼26% for Ni2+ and ∼16% for Cd2+ was observed, suggesting changes in protein conformation upon metal ion interaction. Isothermal calorimetric (ITC) studies show formation of a 1:1 binary complex on interaction with both metal ions but suggest a higher affinity for Ni2+. Fluorescence quenching data suggest a static quenching mechanism of interaction. Various spectroscopic analyses, namely, synchronous fluorescence, ANS fluorescence, far UV CD and ATR-FTIR spectroscopy show that the native mustard cystatin acquires a more ordered conformation upon interaction with metal ions. Differential Scanning Calorimetry indicates that the thermo-stability of the Ni2+ bound protein (Tm=109.4°C) is greater than both, the Cd2+ bound (Tm=104.5°C) and the native (Tm=99.5°C) forms. The B. juncea seed cystatin, is thus, identified as a potent and resilient member of the phytocystatin family with considerable inhibitory capacity despite exposure to heavy metal stress.

Disruption of Energy Transfer and Redox Status by Sulfite in Hippocampus, Striatum, and Cerebellum of Developing Rats.

Patients with sulfite oxidase (SO) deficiency present severe brain abnormalities, whose pathophysiology is not yet elucidated. We evaluated the effects of sulfite and thiosulfate, metabolites accumulated in SO deficiency, on creatine kinase (CK) activity, mitochondrial respiration and redox status in hippocampus, striatum and cerebellum of developing rats. Our in vitro results showed that sulfite and thiosulfate decreased CK activity, whereas sulfite also increased malondialdehyde (MDA) levels in all brain structures evaluated. Sulfite further diminished mitochondrial respiration and increased DCFH oxidation and hydrogen peroxide production in hippocampus. Sulfite-induced CK activity decrease was prevented by melatonin (MEL), resveratrol (RSV), and dithiothreitol while increase of MDA levels was prevented by MEL and RSV. Regarding the antioxidant system, sulfite increased glutathione concentrations in hippocampus and striatum. In addition, sulfite decreased the activities of glutathione peroxidase in all brain structures, of glutathione S-transferase in hippocampus and cerebellum, and of glutathione reductase in cerebellum. In vivo experiments performed with intrahippocampal administration of sulfite demonstrated that this metabolite increased superoxide dismutase activity without altering other biochemical parameters in rat hippocampus. Our data suggest that impairment of energy metabolism and redox status may be important pathomechanisms involved in brain damage observed in individuals with SO deficiency.

Hydrophilic Cu9S5 nanocrystals: a photothermal agent with a 25.7% heat conversion efficiency for photothermal ablation of cancer cells in vivo.

Photothermal ablation (PTA) therapy has a great potential to revolutionize conventional therapeutic approaches for cancers, but it has been limited by difficulties in obtaining biocompatible photothermal agents that have low cost, small size (<100 nm), and high photothermal conversion efficiency. Herein, we have developed hydrophilic plate-like Cu(9)S(5) nanocrystals (NCs, a mean size of ∼70 nm × 13 nm) as a new photothermal agent, which are synthesized by combining a thermal decomposition and ligand exchange route. The aqueous dispersion of as-synthesized Cu(9)S(5) NCs exhibits an enhanced absorption (e.g., ∼1.2 × 10(9) M(-1) cm(-1) at 980 nm) with the increase of wavelength in near-infrared (NIR) region, which should be attributed to localized surface plasmon resonances (SPR) arising from p-type carriers. The exposure of the aqueous dispersion of Cu(9)S(5) NCs (40 ppm) to 980 nm laser with a power density of 0.51 W/cm(2) can elevate its temperature by 15.1 °C in 7 min; a 980 nm laser heat conversion efficiency reaches as high as 25.7%, which is higher than that of the as-synthesized Au nanorods (23.7% from 980 nm laser) and the recently reported Cu(2-x)Se NCs (22% from 808 nm laser). Importantly, under the irradiation of 980 nm laser with the conservative and safe power density over a short period (∼10 min), cancer cells in vivo can be efficiently killed by the photothermal effects of the Cu(9)S(5) NCs. The present finding demonstrates the promising application of the Cu(9)S(5) NCs as an ideal photothermal agent in the PTA of in vivo tumor tissues.

Salt stress induces a decrease in excitation energy transfer from phycobilisomes to photosystem II but an increase to photosystem I in the cyanobacterium Spirulina platensis.

The effects of salt stress (0-0.8M NaCl) on excitation energy transfer from phycobilisomes to photosystem I (PSI) and photosystem II (PSII) in the cyanobacterium Spirulina platensis were investigated. Salt stress resulted in a significant decrease in photosynthetic oxygen evolution activity and PSII electron transport activity, but a significant increase in PSI electron transport activity. Analyses of the polyphasic fluorescence transients (OJIP) showed that, with an increase in salt concentration, the fluorescence yield at the phases J, I and P declined considerably and the transient almost leveled off at 0.8M NaCl. Analyses of the JIP test demonstrated that salt stress led to a decrease in the maximal efficiency of PSII photochemistry, the probability of electron transfer beyond Q(A), and the yield of electron transport beyond Q(A). In addition, salt stress resulted in a decrease in the electron transport per PSII reaction center, but an increase in the absorption per PSII reaction center. However, there was no significant change in the trapping per PSII reaction center. Furthermore, there was a decrease in the concentration of the active PSII reaction centers. Analyses of 77K chlorophyll fluorescence emission spectra excited either at 436 or 580nm showed that salt stress inhibited excitation energy transfer from phycobilisomes to PSII but induced an increase in the efficiency of energy transfer from phycobilisomes to PSI. Based on these results, it is suggested that, through a down-regulation of PSII reaction centers and a shift of excitation energy transfer in favor of PSI, the PSII apparatus was protected from excess excitation energy.

Regulatory subunit I-controlled protein kinase A activity is required for apical bile canalicular lumen development in hepatocytes.

Signaling via cAMP plays an important role in apical cell surface dynamics in epithelial cells. In hepatocytes, elevated levels of cAMP as well as extracellular oncostatin M stimulate apical lumen development in a manner that depends on protein kinase A (PKA) activity. However, neither the identity of PKA isoforms involved nor the mechanisms of the cross-talk between oncostatin M and cAMP/PKA signaling pathways have been elucidated. Here we demonstrate that oncostatin M and PKA signaling converge at the level of the PKA holoenzyme downstream of oncostatin M-stimulated MAPK activation. Experiments were performed with chemically modified cAMP analogues that preferentially target regulatory subunit (R) I or RII holoenzymes, respectively, in hepatocytes. The data suggest that the dissociation of RI- but not RII-containing holoenzymes, as well as catalytic activity of PKA, is required for apical lumen development in response to elevated levels of cAMP and oncostatin M. However, oncostatin M signaling does not stimulate PKA holoenzyme dissociation in living cells. Based on pharmacological and cell biological studies, it is concluded that RI-controlled PKA activity is essential for cAMP- and oncostatin M-stimulated development of apical bile canalicular lumens.

Spectroscopic investigation on the binding of antineoplastic drug oxaliplatin to human serum albumin and molecular modeling.

This study was designed to examine the interaction of oxaliplatin with human serum albumin (HSA) under physiological conditions by using fluorescence, absorption, FT-IR and circular dichroism (CD) spectroscopic techniques in combination with molecular docking study. Spectroscopic analysis of the emission quenching at different temperatures has revealed that the quenching mechanism of oxaliplatin with HSA was static quenching mechanism. The value of 1.64nm for the distance r between the donor (HSA) and acceptor (oxaliplatin) was derived from the fluorescence resonance energy transfer. From the CD and FT-IR results, it was apparent that the interaction of oxaliplatin with HSA caused a conformational change of the protein. Molecular docking study showed that oxaliplatin bind to residues located in subdomain IIA of HSA. The effect of metal ions and amino acids on the binding constant of HSA-oxaliplatin complex was also discussed.

Constitutively-active human LH receptors are self-associated and located in rafts.

Several naturally occurring mutations in human luteinizing hormone receptors (LHR) at position 578 are associated with constitutive activation of the receptor. To determine whether human LHRs that signal in the absence of ligand are self-associated, fluorescence resonance energy transfer (FRET) between receptors was evaluated. Values for FRET between wild type LHR in the absence of ligand were less than 1% and increased significantly to over 11% after exposure to hCG. Constitutively active receptors exhibited 11-15% FRET efficiency in the absence of hormone and these values did not change with hCG treatment. A large fraction of constitutively active LHR-D578H receptors were also associated with so-called plasma membrane rafts. Disruption of these membrane microdomains reduced FRET efficiency but did not affect signalling through cAMP. Thus, in the absence of ligand, constitutively active receptors are self-associated and located in high buoyancy membrane fractions, both characteristics of the hormone-treated wild type receptor.

Nonlinear absorption in biological tissue for high intensity focused ultrasound.

In recent years the propagation of the high intensity focused ultrasound (HIFU) in biological tissue is an interesting area due to its potential applications in non-invasive treatment of disease. The base principle of these applications is the heat effect generated by ultrasound absorption. In order to control therapeutic efficiency, it is important to evaluate the heat generation in biological tissue irradiated by ultrasound. In his paper, based on the Khokhlov-Zabolotkaya-Kuznetsov (KZK) equation in frequency-domain, the numerical simulations of nonlinear absorption in biological tissues for high intensity focused ultrasound are performed. We find that ultrasound thermal transfer effect will be enhanced with the increasing of initial acoustic intensity due to the high harmonic generation. The concept of extra absorption factor is introduced to describe nonlinear absorption in biological tissue for HIFU. The theoretical results show that the heat deposition induced by the nonlinear theory can be nearly two times as large as that predicated by linear theory. Then, the influence of the diffraction effect on the position of the focus in HIFU is investigated. It is shown that the sound focus moves toward the transducer compared with the geometry focus because of the diffraction of the sound wave. The position of the maximum heat deposition is shifted to the geometry focus with the increase of initial acoustic intensity because the high harmonics are less diffraction. Finally, the temperature in the porcine fat tissue changing with the time is predicated by Pennes' equation and the experimental results verify the nonlinear theoretical prediction.

70 microM caffeine treatment enhances in vitro force and power output during cyclic activities in mouse extensor digitorum longus muscle.

Caffeine ingestion by human athletes has been found to improve endurance performance primarily acting via the central nervous system as an adenosine receptor antagonist. However, a few studies have implied that the resultant micromolar levels of caffeine in blood plasma (70 microM maximum for humans) may directly affect skeletal muscle causing enhanced force production. In the present study, the effects of 70 microM caffeine on force and power output in isolated mouse extensor digitorum longus muscle were investigated in vitro at 35 degrees C. Muscle preparations were subjected to cyclical sinusoidal length changes with electrical stimulation conditions optimised to produce maximal work. 70 microM caffeine caused a small but significant increase (2-3%) in peak force and net work produced during work loops (where net work represents the work input required to lengthen the muscle subtracted from the work produced during shortening). However, these micromolar caffeine levels did not affect the overall pattern of fatigue or the pattern of recovery from fatigue. Our results suggest that the plasma concentrations found when caffeine is used to enhance athletic performance in human athletes might directly enhance force and power during brief but not prolonged activities. These findings potentially confirm previous in vivo studies, using humans, which implied caffeine ingestion may cause acute improvements in muscle force and power output but would not enhance endurance.

Radial and temporal variations in surface heat transfer during cryogen spray cooling.

Cryogen spray cooling (CSC) is a heat extraction process that protects the epidermis from thermal damage during dermatologic laser surgery. The objective of the present work is to investigate radial and temporal variations in the heat transferred through the surface of a skin phantom during CSC. A fast-response thermal sensor is used to measure surface temperatures every 1 mm across a 16 mm diameter of the sprayed surface of the phantom. An analytical expression based on Fourier's law and Duhamel's theorem is used to compute surface heat fluxes from temperature measurements. Results show that radial and temporal variations of the boundary conditions have a strong influence on the homogeneity of heat extraction from the skin phantom. However, there is a subregion of uniform cooling whose size is time dependent. It is also observed that the surface heat flux undergoes a marked dynamic variation, with a maximum heat flux occurring at the centre of the sprayed surface early in the spurt followed by a quick decrease. The study shows that radial and temporal variations of boundary conditions must be taken into account and ideally controlled to guarantee uniform protection during CSC of human skin.

Thermal-electrical modeling for epicardial atrial radiofrequency ablation.

Epicardial radiofrequency ablation is increasingly being used for intraoperative treatment of atrial fibrillation. However, the effect of different parameters on the lesion characteristics has not been sufficiently characterized. We used a finite element model to calculate the temperature distribution in the atrial tissue under different conditions during a constant voltage radiofrequency ablation. Our simulation results show that although in the case of a thin atrium the lesion was less deep for a thin atrium, it was easier to achieve transmurality. While considering a thinner atrium, the location of the hottest point of the lesion shifted from the electrode tip to epicardial surface. This effect was due to the convective cooling of the circulating blood inside the atrium. This convective cooling phenomenon has almost negligible effects for atria thicker than 3 mm. The variability of the cooling values has no significant effect on the lesion, even for thin atria (1-2 mm). Increasing the electrode insertion depth (ID) in the tissue produced larger lesions. However, for thinner atria (thickness <2 mm), this increase in the ID reduced the lesion width. It was also proved that the presence of a fat layer between the electrode and the atrial tissue decreased significantly the lesion dimensions.

Interaction of amphotericin B and its low toxic derivative, N-methyl-N-D-fructosyl amphotericin B methyl ester, with fungal, mammalian and bacterial cells measured by the energy transfer method.

Amphotericin B (AMB) derivative, N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME) retains the broad antifungal spectrum and potency of the parent antibiotic, whereas its toxicity towards mammalian cells is reduced by about two orders of magnitude. The purpose of this work was to find out whether the differences observed in the toxicity of MFAME and native AMB are due to the differential drugs affinity to fungal and mammalian cell membranes. Comparative studies on AMB and MFAME biological activity and their affinity to fungal, mammalian and bacterial cells were performed. The interaction of AMB and MFAME with cells have been studied by fluorescence method based on the energy transfer between membrane fluorescent probe (donor) and the polyenic chromophore of the antibiotic (acceptor) simultaneously present in the cell membrane. The amount of the antibiotic bound to cells was indicated by the extent of fluorescence quenching of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) or 1,6-diphenyl-1,3,5-hexatriene (DPH) by polyenic chromophore of the antibiotic. The results obtained indicate that binding extent and characteristics for both antibiotics are comparable in the three types of cells studied. Dramatically lower toxicity of MFAME as compared to AMB towards mammalian cells is not related to the antibiotic-cell affinity, but rather to different consequences of these interactions for cells, reflected in membrane permeabilization. MFAME is definitely less effective than parent AMB in the permeabilizing species formation in mammalian cell membrane.

Molecular proximity of Kv1.3 voltage-gated potassium channels and beta(1)-integrins on the plasma membrane of melanoma cells: effects of cell adherence and channel blockers.

Tumor cell membranes have multiple components that participate in the process of metastasis. The present study investigates the physical association of beta1-integrins and Kv1.3 voltage-gated potassium channels in melanoma cell membranes using resonance energy transfer (RET) techniques. RET between donor-labeled anti-beta1-integrin and acceptor-labeled anti-Kv1.3 channels was detected on LOX cells adherent to glass and fibronectin-coated coverslips. However, RET was not observed on LOX cells in suspension, indicating that molecular proximity of these membrane molecules is adherence-related. Several K(+) channel blockers, including tetraethylammonium, 4-aminopyridine, and verapamil, inhibited RET between beta1-integrins and Kv1.3 channels. However, the irrelevant K(+) channel blocker apamin had no effect on RET between beta1-integrins and Kv1.3 channels. Based on these findings, we speculate that the lateral association of Kv1.3 channels with beta1-integrins contributes to the regulation of integrin function and that channel blockers might affect tumor cell behavior by influencing the assembly of supramolecular structures containing integrins.

Mechanism of pluronic effect on P-glycoprotein efflux system in blood-brain barrier: contributions of energy depletion and membrane fluidization.

Pluronic block copolymer, P85, inhibits the P-glycoprotein (Pgp) drug efflux system and increases the permeability of a broad spectrum of drugs in the blood-brain barrier (BBB). This study examines the mechanisms by which P85 inhibits Pgp using bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of the BBB. The hypothesis was that simultaneous alterations in intracellular ATP levels and membrane fluidization in BBMEC monolayers by P85 results in inhibition of the drug efflux system. The methods included the use of 1) standard Pgp substrate rhodamine 123 to assay the Pgp efflux system in BBMEC, 2) luciferin/luciferase assay for ATP intracellular levels, and 3) 1,6-diphenyl-1,3,5-hexatriene for membrane microviscosity. Using 3H-labeled P85 and fluorescein-labeled P85 for confocal microscopy, this study suggests that P85 accumulates in the cells and intracellular organelles such as the mitochondria where it can interfere with metabolic processes. Following exposure of BBMEC to P85, the ATP levels were depleted, and microviscosity of the cell membranes was decreased. Furthermore, P85 treatment decreased Pgp ATPase activity in membranes expressing human Pgp. A combination of experiments examining the kinetics, concentration dependence, and directionality of P85 effects on Pgp-mediated efflux in BBMEC monolayers suggests that both energy depletion (decreasing ATP pool available for Pgp) and membrane fluidization (inhibiting Pgp ATPase activity) are critical factors contributing to the activity of the block copolymer in the BBB.

Clustering of peptide-loaded MHC class I molecules for endoplasmic reticulum export imaged by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer between cyan fluorescent protein- and yellow fluorescent protein-tagged MHC class I molecules reports on their spatial organization during assembly and export from the endoplasmic reticulum (ER). A fraction of MHC class I molecules is clustered in the ER at steady state. Contrary to expectations from biochemical models, this fraction is not bound to the TAP. Instead, it appears that MHC class I molecules cluster after peptide loading. This clustering points toward a novel step involved in the selective export of peptide-loaded MHC class I molecules from the ER. Consistent with this model, we detected clusters of wild-type HLA-A2 molecules and of mutant A2-T134K molecules that cannot bind TAP, but HLA-A2 did not detectably cluster with A2-T134K at steady state. Lactacystin treatment disrupted the HLA-A2 clusters, but had no effect on the A2-T134K clusters. However, when cells were fed peptides with high affinity for HLA-A2, mixed clusters containing both HLA-A2 and A2-T134K were detected.

A molecular NAND gate based on Watson-Crick base pairing.

[reaction: see text] A DNA-binding dye, 4',6-diamidino-2-phenylindole (DAPI) signals AT base pairing with a shift in the fluorescence emission spectrum. The signaling follows W-C base-pairing rules, and both dAMP and dTMP are required for the largest spectral shift. Thus, the dye with its two phosphate receptor sites functions as a molecular NAND gate accepting nucleotides as inputs. Moreover, when the observation wavelength is changed from 470 to 411.5 nm, the gate functions in TRANSFER logic.

Single-molecule imaging of EGFR signalling on the surface of living cells.

The early events in signal transduction from the epidermal growth factor (EGF) receptor (EGFR) are dimerization and autophosphorylation of the receptor, induced by binding of EGF. Here we observe these events in living cells by visualizing single molecules of fluorescent-dye-labelled EGF in the plasma membrane of A431 carcinoma cells. Single-molecule tracking reveals that the predominant mechanism of dimerization involves the formation of a cell-surface complex of one EGF molecule and an EGFR dimer, followed by the direct arrest of a second EGF molecule, indicating that the EGFR dimers were probably preformed before the binding of the second EGF molecule. Single-molecule fluorescence-resonance energy transfer shows that EGF-EGFR complexes indeed form dimers at the molecular level. Use of a monoclonal antibody specific to the phosphorylated (activated) EGFR reveals that the EGFR becomes phosphorylated after dimerization.

Penicillin biosynthesis: energy requirement for tripeptide precursor formation by delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Acremonium chrysogenum.

In nonribosomal peptide formation by multifunctional enzymes, peptide synthetases catalyze the activation and directed condensation of amino acids. The peptide synthetase involved in penicillin biosynthesis (ACV synthetase) forms the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine from the respective L-amino acids and ATP. So far, the energy requirements for the nonribosomal process have not been clearly established. For ACV synthetase we show that ATP consumption depends on the reaction conditions employed. By simultaneously estimating peptide and AMP production by employing fluorescence detection and UV spectroscopy, respectively, we have determined the energy consumption with high accuracy. Under unfavorable reaction conditions more than 20 mol of ATP are consumed/mol of tripeptide formed, while optimal conditions permit the expected energy requirement of one ATP for each carboxyl group activation, corresponding to three ATP for tripeptide formation. The third ATP is required for the activation of L-valine to maintain the valyl-thioester stage for epimerization and peptide bond formation, and this high-energy bond is sacrificed by hydrolytic removal of the product. No extra energy is required for the directed transport in peptide elongation. Additional energy consumed has been traced to hydrolytic loss of activated intermediates, as has been shown by the analysis of incomplete reaction mixtures.

Prolonged incubation with low concentrations of mercury alters energy transfer and chlorophyll (Chl) a protein complexes in Synechococcus 6301: changes in Chl a absorption and emission characteristics and loss of the F695 emission band.

Synechococcus PCC 6301 cells grown in the presence of low sublethal levels of (about 2 microM) mercury induced alterations in chlorophyll (Chl) a absorption without significant alterations in phycocyanin. Chl a fluorescence emission in Hg(2+)-raised cells showed a large (about 18 nm) blue shift in the peak emission. No major spectral changes in phycobilisome (PBsome) emission characteristic were noticed, indicating major structural alterations in Chl-protein complexes by incubation with Hg2+ ions. Low temperature (77K) emission spectra of cells grown in the presence of Hg2+ showed a loss of the characteristic Chl a emission band at 695 nm (F695), which is known to be linked to photosystem II photochemistry and to originate from the Chl a of core antenna polypeptide CP 47 of photosystem II. The SDS-PAGE polypeptide profile of thylakoids indicates a loss of a polypeptide(s) with a molecular mass between 40 and 60 kDa by Hg2+ incubation of cells. Our results suggest that prolonged incubation of Synechococcus 6301 cells with low concentrations of Hg2+ affects the Chl a spectral properties and the structure of Chl-protein complexes.

Centrosome behaviour and orientation of centrioles under the action of energy transfer inhibitors.

2,4-dinitrophenol, dinitrophenol together with deoxyglucose, sodium azide and ouabain didn't alter cytoplasmic microtubule (MT) network of cultured PK (pig kidney embryo) cells, meanwhile they induced an increase in the average number of pericentriolar satellites and percentage of centrioles with the primary cilium in these cells. Also all drugs studied increase number of MTs attached to and oriented towards the centrosome. Under the action of ouabain the total number of MTs around the centrosome doubled, meanwhile the number of long MTs emanating from the centrosome increased more than 15 times. Under the action of all drugs studied, except sodium azide, the number of maternal centrioles oriented perpendicularly to the substrate surface increased significantly from that in control cells.