ROLE OF ELECTRIC POLARIZATION IN THE THERMOLUMINESCENCE OF CHLOROPLASTS

Abstract Effect of an external electric field on thermoluminescence and thermodepolarization currents were studied in suspensions and dry films of pea chloroplasts. The external electric field was applied either during interrupted heating at a given temperature ("direct effect") or was "stored" in the sample during cooling in the range –25°C and –70°C ("electret effect").
It is shown that in chloroplast suspension both the luminescence burst upon "direct effect" and the new band peaking between –40°C and –50°C induced by the "electret effect" are accompanied by a preferential decrease in the intensity of the thermoluminescence band around +10°C.
A correlation was established between the polarized state of thylakoids and the thermoluminescence burst induced by the external field. In electret samples of chloroplast suspension the intensity of field induced low temperature thermoluminescence band under different experimental conditions varied in parallel with the intensity of thermodepolarization current. In dry films of chloroplasts, in addition to parallel changes in intensities of thermoluminescence and thermodepolarization current, also the peak positions shifted in the same manner when either humidity of samples or temperature of illumination and of application of external field were varied.
We conclude that the polarization state of thylakoid membrane, which can be governed by an external electric field, plays an important role in determining charge stabilization and recombination properties of photosynthetic units. Our results implicate that electric field induces conformational changes in the membranes which increases the frequency factor ascribed to the recombination processes.     

PHOTOREVERSIBLE CHANGES IN pH OF PEA PHYTOCHROME SOLUTIONS

Abstract— Photoinduced pH changes in unbuffered solutions of undegraded pea phytochrome were studied at 10_oC by using a glass electrode. Red light irradiation caused alkalinization of the solutions in the pH range 5.2–xs7.5 and acidification in pH 7.5–8.9. The pH changes were fully reversed by a subsequent irradiation with far-red light. The red and far-red light effects were repeatedly reversible. The solution of tryptic peptide of phytochrome (mol. wt 60000) showed similar photoreversible pH changes.     

EFFECT OF HUMIDITY ON PHOTOINDUCED ESR SIGNAL FROM HUMAN HAIR

Abstract— Human red hairs, equilibrated at 79.8%, 58.0%, 32.3% humidity and at dry conditions, were irradiated with UV-visible (Λ > 290 nm) light. ESR intensity of the photo-induced transient free radicals was measured, at room temperature, as a function of time during and after exposure to light. Photoenhancement was found to be most pronounced in 79.8%-humidity conditioned hair and least in dry hair. The lifetimes of the transient free radicals in these samples were measured as 360s (79.8%), 180s (58.0%), 140s (32.3%), and 50s (dry). The gradual increase in lifetime as a function of humidity conditions was attributed to an increasing number of decay rates of the centers varying from fast to slow, the slow ones being unable to decay unless the fast ones do. This process is best described by a stretched exponential time function, I ( t ) = I _oexp[-( t/t _o)^α].     

OXYGEN CONSUMPTION AND LUMINESCENCE OF ISOLATED Porichthys PHOTOPHORES IN RESPONSE TO ADRENERGIC STIMULATIONS

Isolated photophores of Porichthys maintained in saline at 20_oC, consume oxygen at a mean rate of 115 ± 5 ± 10 ₃ nmol O₂ min₁ ( n = 165).
In the presence of 0.1 and 1 mM adrenalin or noradrenaline the photophore produces a fast peak of light followed by a slow luminescence. A large increase in oxygen consumption occurs prior to the fast light response and it remains slightly above the resting level during the following slow light emission.
When isolated photophores do not produce the fast peak of light(in response to isoprenalin or after treatment with phentolamine), there is only a slight transient increase of oxygen consumption. During the production of the slow luminescence the oxygen consumption remains close to the resting level.
A relationship between the oxygen consumption and the light emission during the fast luminescence has been found: it is suggested that most of the oxygen consumed could be involved in a triggering mechanism of light production.     

ELECTRIC DICHROISM OF PURPLE MEMBRANE SUSPENSIONS

Abstract— Suspensions of purple membranes were exposed to reversing rectangular pulses of relatively low electric field (less than 100V/cm) at various frequencies. Orientation of the membranes was estimated by measuring linear dichroism at 565nm. In the electric field of frequency lower than about 10Hz the purple membranes tended to align perpendicular to the electric field. This orientation was induced mostly by permanent dipole which was perpendicular to the membrane surface. The value of permanent dipole moment was determined to be 140D per protein at pH 7.0, 25°C, in the presence of 5m M phosphate buffer.
In an electric field of frequency more than 100Hz the membrane tended to align parallel to the electric field. Electric polarizability parallel to the membrane surface was estimated to be 1 ± 10⁻¹² cm³ under the same conditions.
Electric dichroism of light-adapted and dark-adapted purple membranes was found to coincide with each other at high frequency. From this result the angle of retinal to the membrane surface was concluded to be the same between light-adapted and dark-adapted bacteriorhodopsins.     

LOSS OF PHOTOREACTIVATION IN UV-IRRADIATED CULTURED FISH CELLS UNDER DIFFERENT CONDITIONS

Abstract— CAF-MM1 cells derived from a goldfish have photoreactivability for the damage induced by ultraviolet light (UV). When UV-irradiated cells were incubated in the dark at 26_AoC, the longest interval in which photoreactivation (PR) was observed (i.e. effective time for PR), measured by colony formation technique, was about 30 h after the UV irradiation. However, if the cells were incubated at 20_AoC, the effective time was prolonged. Since each time appeared to correspond to the doubling time of the cells at each temperature, the loss of photoreactivability is suggested to be closely related to cell growth or progression of cell cycle. The loss of PR was not observed in the cells held in confluence up to 48 h after UV irradiation, in support of the above suggestion. Photoreactivating enzyme in growing CAF-MM1 cells incubated in the dark for 24 h after UV irradiation was shown to be active, so that it is not possible that the cause of the loss of PR is change in the activity of photoreactivating enzyme.     

THE ELECTROPHOTOLUMINESCENCE OF CHLOROPLASTS

Abstract— Delayed light emission emanating from preilluminated chloroplasts can be perturbed with pulsed DC electric fields (200–4000 V cm^-1), The perturbation produces a strong stimulation of chlorophyll luminescence. During the field perturbation the stimulated emission rises to a maximum, typically within 100μs. and then decays. Two kinetic components, R (rapid) and S (slow)†, are distinguished on the basis of their rise and decay times and their field-dependence. The R component increases exponentially at high fields, decays within 100–300μs during the field pulse and collapses with t _1/2= 15 μs at the end of the field pulse. The S component occurs at low fields, exhibits near saturation at 500 V cm^-1, decays with t _1/2 about 3 ms during the field pulse, and collapses with t _1/2= 38μs at the end of the field pulse. Studies using inhibitors, ionophores, electron donors and electron acceptors associate the R component with ion transport processes. The relation to electron transport associated with Photosystem II is discussed.     

MEASUREMENT OF INDUCTION PERIODS IN INHIBITED FREE-RADICAL OXIDATION BY INFRARED CHEMILUMINESCENCE AT 1270 nm

Infrared chemiluminescence at 1.27 µ. appears after the consumption of phenolic inhibitors in solutions of 1-tetralyl hydroperoxide in tert -butylbenzene (BPh), initiated with di- tert -butyl hyponitrite (BHN) at 68_oC. The induction periods with phenols were in excellent agreement with ones calculated from rates of decomposition of the initiator. Di-(2,2,6,6-tetramethyl-4-piperidinyl) sebacate, benzhyd-rol, a derivative of benzhydrol, and di-rerr-butyl ketoxime under these conditions showed no inhibition or a weak effect. Initiated solutions 2.5 mM in p,p'-di- tert -octyldiphenylamine showed no detectable emission at 1270 nm because of efficient quenching of ₁O₂ by diaryl nitroxides formed from the amine, and because the oxidation products of the amine are peroxy scavengers (Thomas, J. R. 1960, J. Amer. Chem. Soc. 82, 5955). The relative yields of IR luminescence from several alkyl hydroperoxides and oxygen-hydrocarbon mixtures, initiated in each case by BHN, were found to vary substantially with the structure of the hydrocarbon and with the solvent.     

ELECTRIC LINEAR DICHROISM OF P700 CHLOROPHYLL U-PROTEIN COMPLEXES

Abstract— The P₇₀₀ chlorophyll a -protein complex (CPI) isolated from green plants was oriented in aqueous solutions using pulsed electric fields of up to 6700 V cm^-1. The electric linear dichroism spectrum is reported in the range of 400–720nm. Positive peaks in the linear dichroism Δ A = A _I - A ₁ (where A_I and A₁ are the absorbance components in which the polarizer orientation is parallel and perpendicular with respect to the electric field. respectively) are observed at 443 and 686 nm. The ΔA signal at 686 nm is discussed in terms of either a specialized chlorophyll form absorbing at 686 nm. or due to an exciton component absorbing at the same wavelength.     

THE EFFECT OF CHRONIC LOW-DOSE UVB RADIATION ON LANGERHANS CELLS, SUNBURN CELLS, UROCANIC ACID ISOMERS, CONTACT HYPERSENSITIVITY and SERUM IMMUNOGLOBULINS IN MICE

Abstract— C3H mice were irradiated three times a week for up to 6 weeks with either 500 J/m² or 1000 J/m² broadband UVB (270–350 nm) or 3000 J/m² narrowband UVB (311–312 nm; TL01 source). Each dose was suberythemal to the mouse strain used. The number of Langerhans cells (LC) in the epidermis was reduced by over 50% after 2 weeks of irradiation with the UVB source and by 20% following TL01 irradiation. Continued irradiation for up to 6 weeks resulted in no further decrease in LC numbers in the case of the UVB source but a steady decline to 40% in the case of the TL01 source. Sunburn cells were detected following irradiation with both sources but the numbers were very low in comparison with acute exposure. Ultraviolet-B exposure resulted in doubling of the thickness of the epidermis throughout the 6 weeks of irradiation while TL01 exposure did not alter epidermal thickness. Conversion of trans- to ew-urocanic acid (UCA) was observed with both UVB and TL01 sources. The percentage of cis -UCA started to return to normal after 4 weeks of TL01 exposure despite continued irradiation. As observed following a single exposure, the contact hypersensitivity (CH) response was significantly reduced following 6 weeks of UVB irradiation but was unaffected by TL01 exposure, indicating no correlation between cis -UCA levels and CH response. Total serum immunoglobulin levels remained unchanged throughout the 6 weeks of UVB or TL01 irradiation but IgE titers significantly increased in all cases in the first 2 weeks of irradiation, indicating a possible shift to a T_H2 cytokine profile. The IgE levels started to return to normal at later times. Thus chronic broadband UVB exposure induces a number of cutaneous and systemic responses that are likely to be dose dependent, while chronic TL0I exposure induces only some of the these responses.     

PHYTOCHROME INTERMEDIATES IN VIVO–II. CHARACTERISATION OF INTERMEDIATES BY DIFFERENCE SPECTROPHOTOMETRY

Abstract— In epicotyl tissue of Pisum , irradiation of P_r at – 196°C forms a stable product P₆₉₈, whereas P_fr forms a stable product P₆₅₀. On warming P₆₉₈, dark transformation to P_r predominates. On warming P₆₅₀ to – 70°C an intermediate P₆₉₀ is formed which bleaches on further warming to –10°C. When tissue is cooled to –196°C during actinic irradiation, difference spectra for subsequent warming to –10°C indicate that P_r, P_fr and an intermediate P₇₁₀ are formed from weakly absorbing intermediates. Complete photoconversion of P_r to P_fr is not possible at temperatures below –5°C. As the temperature is reduced, the amount of P_fr produced from P_r decreases, while P₇₁₀ increases. P₇₁₀ can be photoconverted at –20°C and above, ultimately forming P_r, but in contrast to P_fr it is not photoconvertible at –196°C.     

Photogenotoxicity of Skin Phototumorigenic Fluoroquinolone Antibiotics Detected Using the Comet Assay

Abstract— The fluoroquinolone(FQ) antibiotics photosensitize human skin to solar UV radiation and are reported to photosensitize tumor formation in mouse skin. As tumor initiation will not occur without genotoxic insult, we examined the potential of ciprofloxacin, lomefloxacin, fle-roxacin, BAYy3118 (a recently developed monofluori-nated quinolone) and nalidixic acid to photosensitize DNA damage in V79 hamster fibroblasts in vitro. Cells were exposed to 37.5 kj/m₂ UVA (320-400 nm; glass filtered Sylvania psoralen + UVA (PUVA) tubes; calibrated Waldmann radiometer) at 4A_oC in the presence of FQ and immediately afterwards embedded in agarose, lysed and placed in an electrophoretic field at pH 12. Under these denaturing conditions, the presence of DNA single-strand breaks (SSB), alkali-labile sites (ALS) and double-strand breaks (DSB) can be visualized as DNA migrating away from the nucleus (characteristic "comet" appearance) after staining with a specific fluorochrome. At FQ concentrations that induced minimal loss of cell viability (neutral red uptake assay) the compounds tested induced comets with a rank order of BAYy3118 norfloxacin ciprofloxacin lomefloxacin fleroxacin nalidixic acid. If cells were incubated after treatment for 1 h at 37_oC, the comet score decreased, suggesting efficient removal of SSB/ALS/DSB. Addition of the DNA polymerase, inhibitor, aphidicolin, to cells treated with either ciprofloxacin alone or ciprofloxacin + UVA resulted in an accumulation of SSB due to the endo/exonuclease steps of excision repair. We have demonstrated that the FQ are photogenotoxic in mammalian cells but that FQ-pho-tosensitized SSB are efficiently repaired. Preliminary evidence that ciprofloxacin photosensitizes the formation of DNA lesions warranting excision repair may indicate production of more mutagenic lesions.     

MECHANISM OF ORIENTATION AND LINEAR DICHROISM OF PHOTOSYNTHETIC PARTICLES IN ELECTRIC FIELDS: CHLOROPLASTS AND CHLOROPHYLL-PROTEIN COMPLEXES

Abstract— The mechanisms of orientation in pulsed and alternating electric fields of thylakoids (derived from the sonication of spinach chloroplasts) and of light-harvesting chlorophyll a/b-protein complexes (CPII) were investigated by utilizing linear dichroism techniques. Comparisons of the linear dichroism spectra of thylakoids and CPII particles suggest that the latter are oriented with their directions of largest electronic polarizabilities (and thus probably their largest dimensions) within the thylakoid membrane planes. At low electric field strengths (< 12 V cm^?1), and at low frequencies of alternating electric fields (< 0.25 Hz), thylakoid membranes tend to align with their normals parallel to the direction of the applied electric field; the mechanism of orientation involves a permanent dipole moment of the thylakoids which is oriented perpendicular to the planes of the membranes. However, at high field strengths and high frequencies of the applied alternating electric fields, the thylakoids tend to orient with their planes parallel to the applied field, thus exhibiting an inversion of the sign of the linear dichroism as the electric field strength is increased. At the higher frequencies and at higher field strengths, the orientation mechanisms of the thylakoids involve induced dipole moments related to anisotropies in the electronic polarizabilities. The polarizability is higher within the plane than along a normal to the plane, thus accounting for the inversion of the dichroism as the electric field strength is increased. The CPII particles align with their largest dimension parallel to the applied field at all field strength, indicating that the induced dipole moment dominates the orientation mechanisms in pulsed electric fields. The magnitude of the absolute linear dichroism of CPII suspensions increases with increasing dilution, indicating that aggregates of lower symmetry are formed at higher concentrations of the CPII complexes.     

ELECTRORHEOLOGICAL PROPERTIES OF POLYANILINE/PUMICE COMPOSITE SUSPENSIONS

Electrorheological （ER） properties of polyaniline （PAni）, pumice and polyaniline/pumice composites （PAPC） were investigated. Polyaniline and PAni/pumice composite were prepared by oxidative polymerization. PAni/pumice particlesbased ER suspensions were prepared in silicone oil （SO）, and their ER behavior was investigated as a function of shear rate, electric field strength, concentration and temperature. Sedimentation stabilities of suspensions were determined. It has been found that ER activity of all the suspensions increases with increasing electric field strength, concentration and decreasing shear rate. It has shown that the suspensions have a typical shear thinning non-Newtonian viscoelastic behavior. Yield stress of composite suspensions increased linearly with increasing applied electric field strength and with concentrations of the particles. The effect of high temperature on ER activity of purrfice/silicone oil systems was also investigated.     

FUNCTIONAL ROLE OF CALCIUM IN PHOTORECEPTOR CELLS

Abstract— Ca-uptake by disc membranes prepared from frog rod photoreceptor outer segments was examined. Ca-uptake study revealed two affinity sites which were saturated with 10–⁵ M and 10–³ M of ATP. When disc membranes in 20 m M Tris-HCl buffer (pH 7.5) were stored at -20°C for 6 h, more than 95% of Ca-uptake activity was lost. Ca-uptake activity was, however, preserved if the disc membrane suspension was mixed with 1–10m M ATP and stored at -20°C. Furthermore the reactivation of Ca-uptake was observed if disc membranes, which had lost Ca-uptake ability by storing at 4°C for 3 h, were mixed with 10 m M ATP and then frozen at -20°C for 5 h or 28 h (ATP-induced ATP-dependent Ca-uptake). When the contents of ATP bound to disc membranes were measured during a brief aging at 37°C, the decrement of bound ATP content was correlated well with the decreasing of Ca-uptake activity. Carbonylcyanide m -chlorophenylhydrazone (CCCP), a conductor of protons, inhibited Ca-uptake activity and half maximal inhibition was achieved at 2 × 10–⁸ M. When 10–⁶ M of CCCP was added to the ⁴⁵Ca-accumulated disc membranes, rapid release of ⁴⁵Ca from the disc membranes was observed. These results suggest that ATP may play a role in the Ca-pump regulation in disc membranes and a [H⁺] gradient across disc membrane may be linked to Ca-uptake mechanism.     

SPECTRAL AND TIME DEPENDENCE STUDIES OF THE ULTRA WEAK BIOLUMINESCENCE EMITTED BY THE BACTERIUM Escherichia coli

Abstract— Weak luminescence was detected using photon counting equipment, from oxygenated, liquid cultures of Escherichia coli during two stages of its growth cycle. The first period of emission occurred during the exponential phase of growth and comprised a UV(210–330 nm) band and a visible region(450–620 nm) band, the total intensity being (1.65 ± 0.12) x 10³ counts s^-1. The second period of emission occurred during the stationary phase of growth and comprised only a visible region(450–620 nm) band of intensity (8.72 ± 0.15) x 10³ counts s^-1. When the growth temperature was raised from 306.15 to 310.15 K, the above emission intensities were approximately halved, but the spectra were not changed significantly. No luminescence was observed at either temperature when the E. coli was grown anaerobically. The visible region luminescence was attributed to excited carbonyl groups and excited singlet O₂ dimers formed during the decomposition of lipid peroxides. The UV component was tentatively assigned to oxidative side reactions accompanying the synthesis of proteins.     

Electrical conductivity of graphite suspensions in potassium chloride solutions in direct- and alternating-current electric fields

Electrical conductivity of graphite dispersions in aqueous KCl solutions has been measured. The measurements have been performed in alternating- (1000 Hz) and direct-current electric fields. In an alternating-current electric field, at electrolyte concentrations of 0.0005–0.01 М, the conductivity increases as depending on the mass fraction of the dispersed phase. In 0.1 М solutions, a decrease in the conductivity of the suspension is followed by an increase at dispersed phase contents of higher than 15 wt %. In a direct-current electric field, the conductivity of graphite suspensions (0.001–0.01 М KCl) varies slightly and increases at dispersed phase contents of higher than 15 wt %. In 0.1 М solutions, the specific conductivity of the suspension initially decreases and, then, increases at dispersed phase concentrations above 15 wt %. The unusual electrical properties of the suspensions have been explained as being results of variations in the capacitive and active components of the conductivity of graphite dispersions in electrolytes within the framework of a topological model. Particle polarization and a relatively high capacitive component of the conductivity mainly contribute to an increase in the conductivity of the suspensions in 0.0005–0.01 М electrolytes in the alternating-current electric field. A decrease in the conductivity of suspensions in 0.1 М electrolytes is due to a negative difference between the capacitive and active components of the specific conductivity. It has been assumed that the aggregation of graphite particles yields conducting structures at dispersed phase concentrations above 15 wt %.     

Electrorheological properties of polyaniline suspensions: field-induced liquid to solid transition and residual gel structure

Polyaniline (PANI) was synthesized via oxidative coupling polymerization in acid conditions and de-doped in solution of ammonia. The electrorheological (ER) properties of the PANI/silicone oil suspensions were investigated in oscillatory shear as functions of electric field strength, particle concentration, and host fluid viscosity. Consistent with literature, the PANI ER fluid exhibits viscoelastic behavior under the applied electric field and the ER response is strongly enhanced with increasing electric field strength and particle concentration. The dynamic moduli, G' and G' increase dramatically, by 5 orders of magnitude, as the electric field strength is increased to 2 kV/mm. A viscoelastic liquid to solid transition occurs at a critical electric field strength, in the range Ec = 50-200 V/mm, whose value depends on particle concentration and host fluid viscosity. The fibrillar structure formed in the presence of the applied field has a static yield strength tau(y), whose value scales with electric field strength as tau(y) approximately E(1.88). When the field is switched off a residual structure remains, whose yield stress increases with the strength of the applied field and particle concentration. When the applied stress exceeds the yield stress of the residual structure, fast, fully reversible switching of the ER response is obtained.     

A MICROSECOND KINETIC STUDY OF THE PHOTOGENERATED MEMBRANE POTENTIAL OF BACTERIORHODOPSIN WITH A FAST RESPONDING DYE

Abstract— Bacteriorhodopsin is a light activated proton pump which generates proton and electric gradients across the cytoplasmic membrane of Halobacterium halobium. In this study, a dye whose fluorescence intensity responds rapidly to membrane potential was used to follow the evolution of the potential on liposomes reconstituted with bacteriorhodopsin, in the microseconds time domain. By comparing the formation kinetics of the potential to those of the long-lived intermediate species in the bacteriorhodopsin photocycle, M₄₁₂, both in H₂O and ₂H₂O suspensions, we can draw the following conclusion: the electric potential onset time is 20 μs after initiation of the illumination. The triggering of the potential is not the formation of the M₄₁₂ intermediate, which was hitherto considered to be the first species in the bacteriorhodopsin cycle which has an unprotonated Schiff base linkage at the retinal chromophore. Rather, the potential forms at the transition of the L₅₅₀ intermediate to the species X which precedes M₄₁₂ or even at the preceding conversion of K₅₉₀ to L₅₅₀.     

DYNAMIC FLUORESCENCE OF TRYPTOPHANYL RESIDUES IN LOW MOLECULAR WEIGHT MODEL COMPOUNDS AND PROTEINS

The tryptophanyl fluorescence decay of tuna apomyoglobin, a protein containing a single indole residue, has been compared to that of the monomeric tryptophanyl residue (NATA) in order to discriminate the effect determined by the protein matrix from those induced by physical and chemical agents. The fluorescence decay, obtained in the frequency domain, has been analyzed in terms of lifetime distributions having a Lorentzian shape. The results indicate that the incorporation of the chromophore into the protein matrix determines a broadening of the distribution pattern.
A further increase of the distribution width has been observed on guanidine-induced unfolding of the protein, whereas no effect has been detected for NATA in the presence of guanidine at concentrations as high as 6.0 M. These observations lead to the conclusion that the tryptophanyl fluorescence lifetime is influenced by the protein matrix even in the fully unfolded state.
The increase of temperature from 20 to 45_oC produces an enlargement of the distribution width of NATA and unfolded tuna apomyoglobin. In the same thermal range, the native protein exhibits a narrowing of the tryptophanyl lifetime distribution. This different behaviour has been explained in terms of a larger distribution of microenvironmental states, generated by the chromophore-solvent interaction (which is very limited in the native protein), at higher temperature.
This conclusion has been further corroborated by the observation that a temperature increase changes the weight of the various components which contribute to the total emission of the free amino acid tryptophan.