Membrane Suspensions Research Articles

Action of DCFH and BODIPY as a Probe for Radical Oxidation in Hydrophilic and Lipophilic Domain

Fluorogenic probes such as 2',7'-dichlorofluorescin (DCFH) have been extensively used to detect oxidative events and to measure antioxidant capacity. At the same time, however, the inherent drawbacks of these probes such as non-specificity towards oxidizing species have been pointed out. The present study was carried out to analyze the action and dynamics of 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY) and DCFH as a fluorescent probe in the free radical-mediated lipid peroxidation in homogeneous solution, aqueous suspensions of liposomal membranes and LDL and plasma. The rate constant for the reaction of BODIPY with peroxyl radicals was estimated as 6.0×103 M-1s-1, which makes BODIPY kinetically an inefficient probe especially in the presence of potent radical-scavenging antioxidants such as tocopherols, but a convenient probe for lipid peroxidation. On the other hand, the reactivity of DCFH toward peroxyl radicals was as high as Trolox, a water-soluble analogue of α-tocopherol. Thus, DCFH is kinetically more favored probe than BODIPY and could scavenge the radicals within lipophilic domain as well as in aqueous phase. The partition coefficients for BODIPY and DCFH were obtained as 4.57 and 2.62, respectively. These results suggest that BODIPY may be used as an efficient probe for the free radical-mediated oxidation taking place in the lipophilic domain, especially after depletion of α-tocopherol, while it may not be an efficient probe for detection of aqueous radicals.

Performance evaluation of WDM components based on tunable dielectric membrane technology

Wavelength division multiplexing has become a leading technology for long-haul transmission systems which operate at 1550 nm wavelength. The recent slump in the fibre optics industry has put a strong focus on the development of new flexible and tunable components, as well as cost effectiveness and reliability. Two-chip micromachined devices are very promising candidates to fulfil these demands. A tunable optical filter structure is discussed, which uses a simple bulk-micromachining process based on low-cost dielectric Bragg mirrors. The tuning is achieved by current-induced thermal heating of the membrane suspensions. This concept combines the advantages of low-cost dielectric mirror processing and the simple actuation principle by current flow to create a best-of-breed two-chip solution. The alignment process of the two-chip cavity has been simplified to the point where a simple place-and-fix strategy can be applied. Based on this concept, a tunable two-chip resonant-cavity light-emitting diode (RC-LED) is presented, which utilises the same bulk-micromachined chip. This validates that these micromachined tunable membranes can serve as basic building blocks for a wide variety of tunable components for wavelength division multiplexing systems, such as optical filters, receivers and vertical-cavity surface-emitting lasers (VCSELs).

Partitioning of ρ‐tocopherol in aqueous mixtures of TAG and isolated muscle membranes

AbstractTo study the uptake of ρ‐tocopherol by aqueous suspensions of membranes isolated from chicken muscle and the partitioning of the antioxidant in mixtures of chicken TAG and membranes, ρ‐tocopherol was added to suspensions of these components and they were centrifuged. In an aqueous suspension of membranes only, the tocopherol uptake increased linearly with tocopherol concentration, at about 50% of the added ρ‐tocopherol at all concentrations. The incorporation of tocopherol into the membranes was independent of incubation temperature in the range of 0–37°C. Studies with aqueous mixtures of membranes and TAG suggested a very low exchange of tocopherol between the different lipid fractions upon mixing when the tocopherol resided initially in one lipid fraction. Adding ρ‐tocopherol in ethanol favored incorporation of the antioxidant into the membranes; little antioxidant was incorporated into the membranes when it was added to the suspensions in oil. The results suggest that partitioning of exogenous ρ‐tocopherol between TAG and membrane lipids in muscle foods may be controlled in part by proper selection of the solvent in which it is added.

Bradykinin is not involved in angiotensin converting enzyme modulation of ovarian steroidogenesis and prostaglandin production in frog Rana esculenta

Angiotensin converting enzyme (ACE) was demonstrated to modulate the production of 17beta-estradiol, progesterone and prostaglandin E2 (PGE2) in frog ovary of Rana esculenta. However, the activity was not mediated by angiotensin II (Ang II). In an attempt to identify the peptide involved in the pathway modulated by ACE, bradykinin, another physiological substrate of ACE, was chosen and incubated in the presence of the membrane suspension purified from the frog ovary homogenate. The hydrolytic products were analysed by reverse-phase high-pressure liquid chromatography (HPLC) analysis and the results showed that bradykinin was metabolized by membrane suspension. The presence of the protease inhibitors in the incubation mixture indicated ACE and neutral endopeptidase as being responsible for the bradykinin hydrolysis. Frog ovary was incubated in vitro in the presence of bradykinin (10 microM), bradykinin receptor antagonist NPC 567 (1 mg mL-1), bradykinin fragment (1-7) (10 microM), ACE (2.5 mU mL-1), captopril (0.1 mM) and lisinopril (0.1 mM). The results showed no modulating activity by bradykinin on ovarian 17beta-estradiol and PGE2 production, thus demonstrating that it was not involved in the ACE-modulated pathway.

PH-dependence of electric light scattering by water suspension of purple membranes

The electro-optical behaviour of purple membranes (PM) water suspension in pH range 3.3–6.2 was studied by electric light scattering method. At low degree of orientation the steady-state electro-optical effect (EOE) increases in pH range 3.8–5.6, but an abnormal behaviour of the pH-dependence is observed outside this range. At full orientation EOE has two peaks at pH 3.6 and 5.8, related to changes in membrane geometry. Relaxation time increases in pH range 3.9–5.5, which corresponds to membrane area increase with 20%. The resonance character of pH-dependence around the peaks was explained by reversible alterations in PM curvature, related to asymmetrical increase of the area of both membrane surfaces. © 2002 Elsevier Science B.V. All rights reserved.

Orientation–deformation electro-optical effect in water suspension of purple membranes

Electro-optical methods of light scattering (LS) and dichroism (D) are used to investigate water suspension of purple membranes (PM). The transient processes upon electric pulse turn on and off were measured as well as the field strength dependence of the stationary electro-optic effect (EOE). At fields under 2.5 kV cm − 1 , the PM behaves as rigid particles and the EOE is due to PM orientation. When 5 kV cm − 1 electric pulse is switched off a fast relaxation process of microsecond kinetics and a millisecond relaxation process of disorientation are registered at LS. A process with similar amplitude is absent at D. That fast process is explained with electrically and/or hydrodynamically induced changes in PM form and size, without perceptible changes in membrane internal structure. A conclusion is drawn that the EOE of PM suspension in high electric field has orientation–deformational mechanism. © 2002 Elsevier Science B.V. All rights reserved.

Effect of Red Laser Light on Na+,K+-ATPase Activity in Human Erythrocyte Membranes Sensitized with Zn-Phthalocyanine

The influence of laser light (670 nm) on human erythrocyte membrane Na+,K(+)-ATPase activity in the presence and absence of Zn-phthalocyanine (ZnPc) was studied. The response of erythrocyte membranes to low-power laser irradiation has not been fully elucidated. In our study, we focused on the studies on photo-induced changes of Na+,K(+)-ATPase activity. The erythrocyte membrane suspensions were incubated with 2 mM of ZnPc and next irradiated with energy doses of 19.1, 38.2, 57.3, 76.4, and 95.5 J x cm(-2). The activity of Na+,K(+)-ATPase was assayed colorimetrically at the wavelength of 820 nm and expressed in micromol of inorganic phosphate released from ATP per mg of protein. The measurements of Na+,K(+)-ATPase activity in erythrocyte membranes incubated with ZnPc in the dark demonstrated that all concentrations of the dye (0.5, 1, 2, and 3 microM) stimulated enzyme activity. The concentration of 2 microM caused the smallest increase of enzyme activity, so this concentration was accepted for further studies. Irradiation of erythrocyte membranes in the presence of the dye (2 microM) significantly decreased Na+,K(+)-ATPase activity. Only for energy doses of 19.1 and 38.2 J x cm(-2) was the enzyme activity comparable to the activity of the control. It was found that irradiation with all energy doses applied caused a rise of enzyme activity. In the presence of ZnPc, significant decrease of Na+,K(+)-ATPase activity was observed.

Role of Ca2+-dependent metalloprotease-2 in stimulating Ca2+ ATPase activity under peroxynitrite treatment in bovine pulmonary artery smooth muscle membrane.

We have determined effect of the oxidant peroxynitrite (ONOO-) on Ca2+-dependent matrix metalloprotease-2 (MMP-2) activity and the role of the protease on Ca2+ ATPase activity in bovine pulmonary vascular smooth muscle plasma membrane under ONOO- -triggered conditions. The smooth muscle plasma membrane possesses a 72-kDa protease activity in a gelatin-containing zymogram. The 72-kDa protease activity has been found to be inhibited by tissue inhibitor of metalloprotease-2 (TIMP-2), indicating that the protease is the matrix metalloprotease-2 (MMP-2). Treatment of the membrane suspension with ONOO- caused stimulation of the MMP-2 activity (as evidenced by 14C-gelatin degradation) and also increased Ca2+ ATPase activity. The ONOO- -triggered protease activity and the Ca2+ ATPase activity were found to be inhibited by the antioxidants: vitamin E, thiourea, and mannitol. Pretreatment with catalase and superoxide dismutase did not significantly alter ONOO- -stimulated MMP-2 activity and Ca2+ATPase activity, indicating that peroxide and superoxide are not present in appreciable amount in ONOO-. Under both basal and ONOO- triggered conditions, the MMP-2 activity and the Ca2+ ATPase activity were also inhibited by EGTA, 1:10-phenanthroline, and TIMP-2. However, the ONOO- -stimulated MMP-2 activity and the Ca2+ ATPase activity were found to be insensitive to phenylmethylsulfonylfluoride, Bowman-Birk inhibitor, chymostatin, leupeptin, antipain, N-ethylmaleimide, and pepstatin. These results suggest that ONOO- caused stimulation of MMP-2 activity and that the increased MMP-2 activity subsequently played a pivotal role in stimulating Ca2+ ATPase activity in bovine pulmonary vascular smooth muscle plasma membrane.

Bleaching kinetics of artificial visual pigments with modifications near the ring-polyene chain connection.

Absorbance difference spectra were recorded at 20 degrees C from 30 ns to milliseconds after photolysis of lauryl maltoside suspensions of artificial visual pigments derived from 9-cis isomers of 5-ethylretinal, 8,16-methanoretinal (a 6-s-trans-bicyclic analogue), or 5-demethyl-8-methylretinal. In all three pigments, the earliest intermediate that was detected had the characteristics of a mixture of bathorhodopsin and a blue-shifted intermediate, BSI, which is the first decay product of bathorhodopsin in bovine rhodopsin. The first decays resolved on the nanosecond time scale were the formation of the lumirhodopsin analogues. Subsequent decays were able to be fit with a mechanistic scheme which has been shown to apply to both membrane and detergent suspensions of rhodopsin. Large increases were seen in the amount of metarhodopsin I which appeared after photolysis of 5-ethylisorhodopsin and the bicyclic isorhodopsin analogue, while 5-demethyl-8-methylisorhodopsin more closely followed native rhodopsin in decaying through meta I380, a 380 nm absorbing precursor to metarhodopsin II. In addition to forming more metarhodopsin I, the bicyclic analogue stabilized the metarhodopsin I-metarhodopsin II equilibrium similarly to what has been previously reported for 9-demethylrhodopsin in detergent, introducing the possibility that the bicyclic analogue could similarly be defective in transducin activation. These observations support the idea that long after initial photolysis, structural details of the retinylidene chromophore continue to play a decisive role in processes leading to the activated form, metarhodopsin II.

Role of membrane-associated Ca+ dependent matrix metalloprotease-2 in the oxidant activation of Ca2+Atpase by tertiary butylhydroperoxide.

Treatment of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant tert-butylhydroperoxide (t-buOOH) increases Ca2+ATPase activity. The smooth muscle plasma membrane possesses a Ca2+ dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA and the tissue inhibitor of metalloprotease-2 (TIMP-2). Since 72 kDa is the molecular mass of MMP-2 and since in our present study the 72 kDa protease in the gelatin containing zymogram is inhibited by matrix metalloprotease inhibitors, EGTA and TIMP-2, it may be suggested that the 72 kDa protease is the MMP-2. In addition to the increasing Ca2+ATPase activity, t-buOOH also enhances the activity of the membrane associated Ca2+ dependent protease that degrades 14C-gelatin. The oxidant triggered protease activity and the Ca2+ATPase activity were found to be prevented by the antioxidant vitamin E, and also by the Ca2+ dependent matrix metalloprotease inhibitors: EGTA and TIMP-2. Adding MMP-2 to the smooth muscle plasma membrane suspension caused an increase in Ca2+ATPase activity and pretreatment with TIMP-2 prevents the increase in Ca2+ATPase activity. Combined treatment of the smooth muscle plasma membrane with low doses of MMP-2 and t-buOOH augments further the Ca2+ATPase activity caused by the respective doses of either t-buOOH or MMP-2. Pretreatment with TIMP-2 prevents the increase in Ca2+ATPase activity elicited by the low doses of MMP-2 and/or t-buOOH.

Lanthanide modulation of the orientation of macromolecules induced by purple membrane.

Addition of Tb3+ to purple membrane (PM) suspensions changes the orientation of the menbrane normal from parallel to perpendicular with respect to the magnetic field. Residual dipolar couplings measured in protein L in the presence of PM are scaled by a factor of -1/2. NMR line broadening and cross-correlation effects induced by the addition of PM are partially reversed by Tb3+ but not by Tm3+ that has no effect on the orientation of PM. This is interpreted as the result of anisotropically restricted motion of protein L in the transiently PM-bound form.

Bacteriorhodopsin: a natural, efficient (nonlinear) photonic crystal

From the angular dependence of the second-order nonlinear light scattering (hyper-Rayleigh scattering) from a suspension of purple membrane, bacteriorhodopsin was recently shown to exhibit (nonlinear) photonic crystal properties [Opt. Lett.25, 1391 (2000)]. The optical nonlinearity, i.e., the first hyperpolarizability β, is localized in the small retinal moiety, whereas the optically linear refractive index n is relevant to the large membrane protein. The combination of the nonlinear hyperpolarizability of the retinal decoupled from the linear refractive index of the protein explains the observed angular dependence. The temporal evolution of this angular dependence has now been analyzed. The disappearance of the angular dependence of the nonlinear scattering is shown to be a consequence of the solubilization of the large purple membrane patches into individual protein monomers. This result strongly suggests that decoupling of the optical nonlinearity from the phase-matching condition for coherent second-harmonic generation will result in highly efficient coherent second-harmonic generation in bacteriorhodopsin crystals. In addition, a simulation of the bandgap properties was made for an abstracted structure with a large refractive index for the retinal and with a small refractive index for the protein matrix.

Coating and fusing cell membranes onto a silica surface and their chromatographic characteristics

A new cell membrane stationary phase (CMSP) consisting of porous silica coated with active cell membranes is presented for affinity chromatography. By immersing, silica into a suspension of cell membranes, the whole surface of silica was covered by the cell membranes due to the irreversible adsorption of silanol groups (Si−OH) on the silica surface and the self-fusion of the cell membranes. CMSP can be used directly as a chromatographic packing material without any additional chemical modification. The surface characteristics, enzymatic activity, and chromatographic behavior of CMSP were investigated. The results obtained from scanning electron microscope, surface energy spectrometer, enzyme assay, and liquid chromatography showed that the surface characteristics of CMSP were very different from that of normal and reversed stationary phases. CMSP was found to have the characteristics of both cell membrane activity and chromatographic separation. Moreover, CMSP, as a chiral stationary phase, could be used for the enantiomeric separation of (±) Bay-K8644. The capacity factor of some calcium antagonists on CMSP was found to have a good correlation with their pharmacological actions. It is concluded that CMSP may be used not only as a kind of packing material in bio-affinity chromatography, but also as a tool for studying the interactions between a drug and its receptor.

Local coherence and the temporal development of secondharmonic emission

In a variety of mesoscopically disordered systems, high levels of opticalexcitation resulting from pulsed laser irradiation can establish opticalcoherence within separate particles or locally ordered domains, leading tosecond harmonic emission whose temporal signature characterizes thedecay of the excited state population. Examples of such systems willinclude colloids, cell and membrane suspensions, and many plastics,glasses and other modern materials. With pulsed excitation of sufficientintensity to elicit the onset of saturation, second harmonic emission onthe throughput of a subsequent probe beam exhibits a characteristic decayand recovery. Detailed calculations show that such features arise not onlyin systems whose optical response involves two discrete levels, but alsoin systems of considerably greater electronic complexity. Deconvolutionof the temporal trace of the harmonic signal serves as an independentmeans of monitoring the decay of the excited state. The extent of recoveryin the harmonic signal also serves to register the extent of localcoherence, and hence in many systems the localization of structural order.Finally, the principles introduced in the theory are shown to be applicableto other types of system such as certain photochromic materials.

Effect of Li+ upon the Mg2+-Dependent Activation of Recombinant Giα1

Although lithium salts have been used in the treatment and prophylaxis of manic-depressive or bipolar patients for 50 years, the mechanism of the pharmacologic action of Li+ is unknown. Based on activity studies of inhibitory and stimulatory guanine-binding (G) proteins in rat cortical membranes, it was proposed that Li+ inhibition of G-proteins may account for its pharmacologic action. We used the purified α subunit of the recombinant inhibitory G-protein, rGiα1, and found that Li+ at therapeutic levels significantly inhibited the formation of the GDP.AlF−4.rGiα1 complex. Because our studies were conducted with a purified, metal-reconstitutedG-protein rather than with cell membrane suspensions, our Li+ inhibition results lend additional support to the G-protein hypothesis for Li+ action.

Bulk micromachined electrostatic RMS-to-DC converter

Bulk micromachining in silicon and glass wafers and subsequent silicon-to-glass anodic bonding have been used for the realization of an electrostatic RMS-to-DC converter. A suspended membrane has been designed for: large dynamic operating range (detection limit by minimum mechanical-thermal noise and high value of the pull-in voltage), maximum bandwidth (low series resistance, high second harmonic suppression using squeeze film damping and suspension beam design), long-term stability, and a sufficient displacement-to-voltage sensitivity (membrane area and suspension arm length). Prototypes are typically composed of a 3 /spl times/ 3 mm/sup 2/ perforated membrane area suspended by four beams of 200 /spl mu/m length, 500 /spl mu/m width, and 4 /spl mu/m thickness micromachined out of silicon and aligned to a counter electrode on glass with 4-/spl mu/m spacing in between. Measurements on realized devices show a 4.5 pF nominal capacitance. Static measurements indicate a sensitivity of 5 fF/V/sup 2/ and a voltage shift of 0.2 V. The nominal square relation is achieved within a 0.5% nonconformity error.

Rat cerebral cortex corticotropin-releasing hormone receptors: evidence for receptor coupling to multiple G-proteins.

The wide distribution of corticotrophin-releasing hormone (CRH) receptors in brain and periphery appear to be important in integrating the responses of the brain, endocrine and immune systems to physiological, psychological and immunological stimuli. The type 1 receptors are highly expressed throughout the cerebral cortex, a region involved in cognitive function and modulation of stress responses, where they are coupled to the adenylyl cyclase system. Using techniques that analyse receptor-mediated guanine-nucleotide binding protein (G-proteins) activation, we recently demonstrated that expressed type 1alpha CRH receptors are capable of activating multiple G-proteins, which suggests that CRH can regulate multiple signalling pathways. In an effort to characterize the intracellular signals generated by CRH in the rat cerebral cortex we sought to identify G-proteins activated by CRH in a physiological membrane environment. Rat cerebral cortical membrane suspensions were analysed for the ability of CRH to stimulate incorporation of [alpha-32P]-GTP-gamma-azidoanilide to various G-protein alpha-chains. Our results show that CRH receptors are coupled to and activate at least five different G-proteins (Gs, Gi, Gq/11, Go and Gz) with subsequent stimulation of at least two intracellular signalling cascades. In addition, the photoaffinity experiments indicated that the CRH receptors preferentially activate the 45 kDa form of the Gs alpha-protein. This data may help elucidate the intracellular signalling pathways mediating the multiple actions of CRH especially under different physiological conditions.

Effect of NADPH on formation and decay of human metarhodopsin III at physiological temperatures

Difference absorption spectra were recorded during the formation and decay of metarhodopsin III after sonicated membrane suspensions of rhodopsin were bleached at 37°C. The data were analyzed using SVD, spectral decomposition and global exponential fitting. By comparison of the results in the presence or absence of 70 μM NADPH and those for bovine or human rhodopsin, a single comprehensive scheme was fit to all the data, including reduction of retinal to retinol by the intrinsic retinol dehydrogenase. On the time scale studied the mechanism involves two 382 nm absorbing species and two 468 nm, absorbing species, supporting the notion that human metarhodopsin III is not a homogeneous species. The results confirm that metarhodopsin III forms and persists sufficiently long in the human retina under physiological conditions that it could undergo secondary photoisomerization.

Evidence for Post-translational Membrane Insertion of the Integral Membrane Protein Bacterioopsin Expressed in the Heterologous Halophilic Archaeon Haloferax volcanii

The gene coding for the integral membrane protein bacterioopsin (Bop), that is composed of seven transmembrane helices, was expressed in the halophilic archaeon Haloferax volcanii as a fusion protein with the halobacterial enzyme dihydrofolate reductase and with the cellulose binding domain of Clostridium thermocellum cellulosome. In each case, bacterioopsin was present both in the membrane and in the cytoplasmic fractions. Pulse-chase labeling experiments showed that the fusion protein in the cytoplasmic fraction is the precursor of the membrane-bound species. Bacterioopsin mutants that lack the seventh helix (BopDelta7) were found to accumulate only in the cytoplasmic fraction, whereas bacterioopsin mutants that lack either helices four and five (BopDelta4-5), or helices one and two (BopDelta1-2), were found in the cytoplasmic as well as in the membrane fractions. The seventh helix, when expressed alone, could target in trans the insertion of a separately expressed bacterioopsin mutant protein that has only the first six helices. These results support a model in which bacterioopsin is produced in H. volcanii as a soluble protein and in which its insertion into the membrane occurs post-translationally. According to this model, membrane insertion is directed by the seventh helix.

Vacuolar-type ATPase in the accessory boring organ of Nucella lamellosa (Gmelin) (Mollusca : Gastropoda): role in shell penetration.

The structure and function of the accessory boring organ (ABO) of muricid gastropods has been described in numerous studies, and the ABO of Nucella lamellosa was found to be similar to those of other muricid species. The active cap region of the ABO is composed of tall, mitochondria-rich cells with distinct brush borders at their apicies, surrounding a hemolymph-containing central sinus. Using antibodies specific for vacuolar-type ATPase (V-ATPase), enzyme immunoreactivity was found to be limited to the brush border of the epithelial cells. Electron immunohistochemistry revealed that V-ATPase immunoreactivity resides in the plasma membranes of the microvilli. Immunodot blotting using yeast V-ATPase as a positive control confirmed the specificity of the reactions. SDS-PAGE of membrane suspensions from the ABO revealed protein bands of the requisite molecular weight for V-ATPase subunits. Western blots suggest that antibodies raised against mammalian V-ATPase subunits recognize subunits of the molluscan V-ATPase. The molecular weights of these identified subunits are similar to those in mammals. The V-ATPase-specific inhibitor bafilomycin A1 inhibited ATPase activity in samples of ABO homogenate by about 10% relative to control, providing further evidence for the presence of V-ATPase. Specific V-ATPase activity was about 67 picomoles of inorganic phosphate per microgram of protein per minute in the homogenate. Collectively this evidence strongly suggests that a vacuolar-type proton transporting ATPase is present in the brush border of the accessory boring organ of Nucella lamellosa, and is responsible for acidifying secretions from this gland. Similarities between the ABO, osteoclasts, and the mantle of freshwater bivalves also suggest that the mechanism for decalcification of calcareous substrates is conserved.