Circular dichroism of squid rhodopsin and its intermediates

Circular dichroism (CD) and absorption spectra of squid (Todarodes pacificus) rhodopsin, isorhodopsin and the intermediates were measured at low temperatures. Squid rhodopsin has positive CD bands at wavelengths corresponding the - and β-absorption bands at liquid nitrogen temperature (CD maxima: 485 nm at -band and 348 nm at β-band) as well as at room temperature (CD maxima: 474 nm at -band and 347 nm at β-band). The rotational strength of the -band has a molecular ellipticity about twice that of cattle rhodopsin. The CD spectrum of bathorhodopsin displays a negative peak at 532 nm, the rotational strength of which has an absolute value slightly larger than that of rhodopsin. The reversal in sign at -band of the CD spectrum may indicate that the isomerization of retinal chromophore from twisted 11-cis form to twisted 11-trans form has occurred in the process of conversion from rhodopsin to bathorhodopsin. Lumirhodopsin has a small negative CD band at 490 nm, the maximum of which lies at 25 nm shorter wavelengths than the absorption maximum (515 nm), and a large positive CD band near 290 nm, which is not observed in rhodopsin and the other intermediates. This band may be derived from a conformational change of the opsin. In the process of changing from lumirhodopsin to LM-rhodopsin, the CD bands at visible and near ultraviolet regions disappear. Both alkaline and acid metarhodopsins have no CD bands at visible and near ultraviolet regions.     

Influence of UV-B radiation on photosynthetic light-response curves, absorption spectra and motility of four phytoplankton species

Several unicellular algae were exposed to artificial UV-B (280–320 nm) radiation after adaptation to high (43 W m⁻²) and low (19 W m⁻²) visible light. UV-B radiation had different effects on rates of photosynthesis, motility and absorption spectra for these species. Photosynthesis of Euglena gracilis and the diatom Phaeodactylum tricomution was more sensitive to UV-B inhibition than that of the dinoflagellates Heterocapsa triquetra and Prorocentrum minimum . Not only UV-B radiation but also high visible light had a photoinhibitory effect on photosynthesis in all four organisms. The effect on photosynthesis was observed both on the quantum yield and on the light saturation rate of photosynthesis. The dinoflagellates, in contrast to E. gracilis and P. tricorntum , absorbed strongly in the UV region (334 nm) and their absorption peaks increased after growth under high visible light or with or without UV-B radiation for one week. The swimming speed of H. triquetra decreased more after low visible light and UV-B radiation compared to high visible light and UV-B radiation. The negative effects of UV-B radiation on P. minimum and E. gracilis were most pronounced after high visible light.     

Effects of UV-A (320 to 399 Nanometers) on Grazing Pressure of a Marine Heterotrophic Nanoflagellate on Strains of the Unicellular Cyanobacteria Synechococcus spp

In the open ocean, where turbidity is very low, UV radiation may be an important factor regulating interactions among planktonic microorganisms. The effect of exposure to UV radiation on grazing by a commonly isolated marine heterotrophic nanoflagellate, Paraphysomonas bandaiensis, on two strains of the cyanobacteria Synechococcus spp. was investigated. Laboratory cultures were exposed to a range of irradiances of artificially produced UV-B (290 to 319 nm) and UV-A (320 to 399 nm) for up to 10 h. At a UV-B irradiance of 0.19 W m, but not 0.12 W m, grazing mortality of Synechococcus spp. and nanoflagellate-specific grazing rates were reduced compared to mortality and grazing rates with UV-A treatment. Within 6 h of exposure, UV-A alone suppressed grazing mortality at irradiances as low as 3.02 W m. The extent to which grazing mortality and nanoflagellate-specific grazing rates were suppressed by UV-A increased with both irradiance and duration of exposure. Over a 6-h exposure period, differences in grazing mortality were largely attributable to differential survival of nanoflagellates. Over a longer period of exposure, there was impairment by UV-A alone of nanoflagellate-specific grazing rates. Rates of primary productivity of Synechococcus spp. were also reduced by UV-A. The extent to which Synechococcus productivity was reduced, compared to the reduction in Synechococcus grazing mortality, depended on the duration of UV-A exposure. These results support the hypothesis that UV-A alone influences the composition and biomass of marine microbial communities by affecting predator-prey interactions and primary production.     

A novel UV laser-induced visible blue radiation from protein crystals and aggregates: scattering artifacts or fluorescence transitions of peptide electrons delocalized through hydrogen bonding?

Proteins lacking prosthetic groups and/or cofactors are known to undergo electronic excitation transitions only upon exposure to UV-C (< 280 nm) and UV-B (280-320 nm), but not UV-A (320-400 nm) photons. Here, we report the discovery of a novel excitation that peaks at approximately 340 nm and yields visible violet-blue radiation with apparent band maxima at approximately 425, 445, 470, and 500 nm. All proteins and large polypeptides examined in solid form, and in solutions, display this quenchable and photobleachable radiation which can be established not owing to aromatic sidechains. As a note of caution, we wish to state that we have not been able to completely eliminate the possibility that the radiation may be an artifact owing to second order effects such as, e.g., Raman scattering of Raman-scattered photons; however, we assert that all our experiments indicate that the radiation actually owes to some form of fluorescence. We propose that peptide electrons that have been delocalized through intramolecular or intermolecular hydrogen bond formation display these long-wavelength electronic transitions. If confirmed by future studies, this preliminary discovery may turn out to have important implications for biomolecular spectroscopy, protein crystallography, and materials science.     

Functional phycobilisome core structures in a phycocyanin-less mutant of cyanobacterium Synechococcus sp. PCC 7942

We have constructed a mutant Synechococcus sp. PCC 7942, termed R2HECAT, in which the entire phycobilisome rod operon has been deleted. In the whole cell absorption spectra of R2HECAT, the peak corresponding to phycocyanin (PC), max620 nm, could not be detected. However, a single pigment-protein fraction with max=654 nm could be isolated on sucrose gradients from R2HECAT. Analysis of this pigment-protein fraction by non-denaturing PAGE indicates an apparent molecular mass of about 1200–1300 kDa. On exposure to low temperature, the isolated pigment-protein complex dissociated to a protein complex with a molecular mass of about 560 kDa. When analysed by SDS-PAGE, the pigment-protein fraction was found to consist of the core polypeptides but lacked PC, 27, 33, 30, and the 9 kDa polypeptides which are a part of the rods. All the chromophore bearing polypeptides of the core were found to be chromophorylated. CD as well as absorption spectra showed the expected maxima around 652 and 675 nm from allophycocyanin (APC) and allophycocyanin B (APC-B) chromophores. Low temperature fluorescence and excitation spectra also showed that the core particles were fully functional with respect to the energy transfer between the APC chromophores. We conclude that PC and therefore the rods are dispensable for the survival of Synechococcus sp. PCC 7942. The results indicate that stable and functional core can assemble in absence of the rods. These rod-less phycobilisome core is able to transfer energy to Photosystem II.Abbreviations PS II Photosystem II - PC phycocyanin - APC allophycocyanin - APC-B allophycocyanin B - PAGE polyacrylamide gel electrophoresis - Cml chloramphenicol - kbp kilobase pairs     

Characterization of a dissimilatory-type sulfite reductase, desulfoviridin, from Desulfovibrio africanus Benghazi

A desulfoviridin-type sulfite reductase having the alpha band at 638 nm was purified from Desulfovibrio africanus Benghazi (NCIB 8401) by chromatography on DEAE-cellulose, Sephadex G-200, and DEAE-Sepharose columns and by disc gel electrophoresis. The content of desulfoviridin in the soluble protein was estimated to be about 6% from the purification indexes. Like the typical desulfoviridin from D. vulgaris Miyazaki K, it formed mainly trithionate besides thiosulfate and sulfide in sulfite reduction coupled to hydrogenase and methyl viologen. No significant differences in the amino acid compositions, CD patterns in the UV (205-250 nm) region, and subunit structures were found, except for a pI value about 1 unit larger (pI 5.3). The split Soret (410 +/- 2 nm, less intense peak at 391 +/- 2 nm with a shoulder around 380 nm) and beta (584 +/- 2 nm) band maxima of the enzyme as isolated, and the visible absorption and fluorescence spectra of the acidic acetone-extracted chromophore were almost identical to those ascribed to sirohydrochlorin in spite of the reported difference in the native enzyme (alpha band maxima at 638 nm as against 628 +/- 2 nm in a typical desulfoviridin). Iron was the only significant chelatable metal contained in the chromophore. Some differences between africanus and vulgaris desulfoviridins were observed in the CD patterns in the UV to near UV region (250-340 nm) and also in the visible absorption spectra in the presence of dithionite.     

Epidermal transmittance and phenolic composition in leaves of atrazine-tolerant and atrazine-sensitive cultivars of Brassica napus grown under enhanced UV-B radiation

Experiments were conducted on the atrazine-tolerant mutant Stallion and the atrazine-sensitive cv. Paroll of Brassica napus L., which were grown under either visible light or with the addition of UV-B radiation (280–320 nm) for 15 days. The mutant has been shown to be sensitive to high levels of visible light as compared to the atrazine-sensitive cultivar and therefore we wished to determine plant response to UV-B radiation with respect to potential pigment changes, certain anatomical features, radiation penetration and partial photosynthesis. With regard to pigment changes, we were particularly interested in whether the compositional shift in flavonol pigments under enhanced UV-B radiation, previously suggested to favour increased antioxidant activity, is confined to the adaxial epidermis, which generally receives most UV-B radiation or whether the pigment shift is also inducible in the abaxial epidermis.As was to be expected, the penetration of UV-B radiation (310 nm) was lower in the UV-B-exposed plants, which was correlated with an increased amount of UV-screening pigments in the adaxial and abaxial epidermal layers. The main flavonoid glycosides showed the largest shift from kaempferol to quercetin as aglycone moiety in the adaxial epidermal layer. However, in the abaxial epidermal layer the hydroxycinnamic acid (HCA) derivatives and kaempferol glycosides were predominant. Penetration of 430 nm light was higher after UV-B exposure, and probably contributed to the fact that photosynthetic efficiency of photosystem II was unchanged or higher after UV-B exposure. UV-B radiation decreased leaf area in the atrazine-tolerant mutant only. Both cultivars showed an increased leaf thickness after UV-B exposure due to cell elongation mainly of the palisade tissue. This was especially evident in the mutant.     

Chiral discrimination of 5,6-epoxy-3-dehydroretinal by aporetinochrome and cattle opsin

The combination of a racemic all-trans 5,6-epoxy-3-dehydroretinal (EDR) with aporetinochrome formed a mixture of two diastereomeric pigments. One of the diastereomeric pigments which contained the all-trans EDR with a negative circular dichroic (CD) band, hereafter called EDR(-)-chrome, has its visible absorption maximum around 438 nm, while the other pigment, called EDR(+)-chrome, has its maximum at 464 nm. These were substantiated by measuring the optical activities of the two EDR isomers which were extracted from a mixture of racemic all-trans EDR and a smaller amount of aporetinochrome following exposure to orange light (greater than 530 nm) that irradiates EDR(+)-chrome selectively. The extracted all-trans EDR had a negative CD band around 240 nm and the extracted 11-cis EDR had a positive band in that region with two negative bands on either side of the main band. In the case of both pigments, the effect of alkalinization on the increase of absorbance in the near-ultraviolet and the decrease of absorbance in the visible region was proportionate, qualitatively, to that on the positive CD intensities in both regions. These results suggest that the chromophore EDR in each pigment binds to the same binding site via a Schiff base. The EDR(+)-chrome exhibited properties similar to those of retinochrome, but EDR(-)-chrome showed some different properties, i.e. its formation rate was slower than that of the former one and its absorption band in the near-ultraviolet appeared even at neutral pH. Moreover, by exposing EDR(-)-chrome to yellow light (greater than 480 nm), only a part of its prosthetic all-trans EDR was isomerized and resulted in the formation of 11-cis and 13-cis isomers. This variation in photoisomerizing activity was supposed to be due to the difference in conformation of the side chain between EDR(+) and EDR(-) in aporetinochrome. Combination of 11-cis EDR with cattle opsin was also shown to result in the formation of two diastereomeric pigments. The absorption maxima of the diastereomers containing 11-cis EDR(+) and EDR(-) were at about 446 and 474 nm, respectively.     

Studies on spectral characteristics of allophycocyanin isolated from Anabaena cylindrical: Curve-fitting analysis

Spectroscopic characteristics of allophycocyanin (APC) isolated from Anabaena cylindrica were studied by curve-fitting analyses of absorption spectra measured at room temperature. Gaussian curve-fittings based on the least-squares method were done on absorption spectra for three different dissociation-association conditions of the protein. The composition of the component bands was similar in the three absorption spectra and could be classified into three groups depending on changes in band intensity; the first group was represented by the main band at 653 nm, which was intense in the trimer APC but markedly reduced in the monomer. The second group was represented by the main band at 607 nm, which showed opposite changes. The third group had its main band at 623 nm, and it remained the same in the three absorption spectra. The sum of the band area of the variable components (the first and second groups) was almost equal to that of the constant components (the third group). These results indicate that: (i) APC has two types of phycocyanobilin (PCB) which differ in their spectroscopic characteristics. (ii) The PCB responsible for the variable component bands, and thus for the spectral changes, has two excitation states corresponding to 653 and 607 nm, respectively, and the spectral changes are ascribable to variations in the relative frequencies of occurrence of the two states: in associated APC the first state predominates, and in dissociated APC, the second. (iii) PCB for the constant component bands has only one excitation state corresponding to the 623-nm band. Changes in the emission spectra correlate with these changes in the absorption spectra, suggesting that in APC the phycocyanobilins responsible for and independent of absorption spectrum changes act as the fluorescing and sensitizing types, respectively.     

Effects of UV-B radiation on growth rates,pigment content and ultrastructure of red (wild type), greenish-brown and green strains of Gracilaria birdiae (Gracilariales,Rhodophyta)

Based on physiological characteristics, we hypothesized that different strains of Gracilaria birdiae from two distinct geographical areas of the Brazilian coast (2500 km apart) would have different responses to long-term exposure to UV-B radiation (UV-B). The locations differ in their environmental conditions: one is a warmer area, Ceará State (CE), closer to the equator; the other is a colder area, Espirito Santo State (ES), closer to the Tropic of Capricorn. To test the hypothesis that the CE population is more resistant to UV-B than the ES population, apical segments of the red (RD_CE, RD_ES), green (GR_CE) and greenish-brown (GB_CE) strains were cultivated in the laboratory under two treatments: control (PAR) and artificial UV-B (PAR + UV-B). Algal performance was evaluated by considering growth rates, pigment content and ultrastructural analysis. Compared with the control, all strains showed a decrease in growth rates after exposure to UV-B. Of all strains, RD_ES showed the greatest sensitivity to UV-B. However, a decrease in growth rate and morphological changes were observed to a lesser extent in the RD_CE strain. Moreover, exposure to UV-B resulted in a decrease in the concentrations of phycobiliproteins in the RD_CE strain. The GB_CE strain showed an increase in phycoerythrin (PE)/allophycocyanin (APC) and phycocyanin (PC)/allophycocyanin (APC) ratios after exposure to UV-B, suggesting this strain had a higher tolerance to the radiation. No differences in the chlorophyll a and carotenoid content were found between the control and UV-B treated samples for all strains. Ultrastructural changes, such as damage to chloroplasts and mitochondria, were present in all strains after exposure to UV-B. In summary, our findings support the hypothesis that the population from Ceará State has adapted to the higher irradiation and is thus more resistant to increased UV-B. Additionally, of the strains tested, the GB_CE and RD_CE strains appear to be more resistant to this radiation.     

Protective mechanisms and acclimation to solar ultraviolet-B radiation in Oenothera stricta

Abstract Mechanisms of plant protection and acclimation to potentially damaging solar ultraviolet-B (UV-B, 280–320 nm) radiation incident on the Earth's surface were examined in Oenothera stricta. Attenuation of this radiation in the upper leaf epidermis reduces the penetration of UV-B radiation to the mesophyll where damage to physiologically sensitive targets can occur. The epidermis is a highly selective radiation filter that can attenuate up to 95% of the incident UV-B radiation and yet transmit between 70% and 80% of the visible radiation. Exposure to UV-B radiation significantly reduced the degree of epidermal UV-B transmittance by as much as 33%. No significant reduction in epidermal transmittance of visible radiation was observed as a result of UV-B exposure. The plasticity in epidermal UV-B transmittance results from production of flavonoid and related phenolic compounds in the tissue. Absorbance of UV-B radiation in llavonoid extract solutions from epidermal and mesophyll tissues significantly increased by as much as 100% and 35%, respectively, after exposure to UV-B radiation. Photosynthetic rates of leaves exposed to UV-B radiation were not significantly reduced at dose rates representative of the radiation flux found in the habitat of this species, but significant photosynthetic depression was observed at dose rates that exceed the field UV-B flux. The phenotypic plasticity in epidermal UV-B transmittance resulting in decreased penetration of damaging UV-B radiation to the mesophyll may reduce the rate of damage to a level where repair mechanisms can keep pace with reduced injury.     

Phycoerythrin synthesis is induced by solar UV-B in the cyanobacterium Nostoc

Solar UV-B (280–315 nm) induces the synthesis of phycoerythrin (PE) in a Nostoc species isolated from the Andean high altitude lake Yanaqocha. The outdoor experiments were carried out in a small lake in Erlangen, Germany, using natural conditions. After 2- and 4-h exposure to solar radiation, the immunodetection signal using monoclonal antibodies anti-PE was lower in control cells (exposed to PAR + UV-A) than in cells exposed to total solar radiation (PAR + UV-A + UV-B). Cells exposed at depths in which no UV-B penetrated showed no differences from control cells regarding PE content. When exposed to monochromatic radiation of 280, 300 or 360 nm, purified PE was photodegraded in a wavelength dependent manner resulting in different polypeptide fragments carrying chromophore groups. Immunodetection revealed active synthesis of PE in parallel to photodamage by solar UV-B indicating that PE is important for photoadaptation to shorter wavelengths in the cyanobacterium Nostoc sp.     

Interpretation of the absorption and circular dichroic spectra of oriented purple membrane films.

The absorption and circular dichroic (CD) spectra of purple membrane films in which the plane of the membranes is oriented perpendicular to the incident beam are compared with the solution spectra. This enables one to relate structural features of the purple membrane to a coordinate system as defined by a normal to the membrane plane and two mutually perpendicular in-plane axes. The film and solution absorption spectra were similar except for a relative depression in the 200 - 225-nm region of the film spectrum. However, the CD spectra showed significant differences in the visible region, where the biphasic band in the solution spectrum was replaced by a single positive band at 555 nm in the film spectrum and in the far ultraviolet region, where the 208-nm band was deleted from the film spectra of the native and regenerated membranes. Moreover, a small shoulder occurred at 208 nm in the film spectrum of the bleached membrane. The near ultraviolet spectra also showed differences, whereas the 317-nm band remained essentially the same for both spectra. Based on excitonic interpretations of the visible and far ultraviolet spectra the following conclusions were reached: (a) a relatively strong in-plane monomeric interaction occurs between te retinyl chromophore and apoprotein; (b) the helical axes of the native and regenerated membrane proteins are oriented primarily normal to the membrane plane; and (c) the helical axes of the bleached membrane proteins are tilted more in-plane than the axes of the native or regenerated membrane. Additional conclusions were that an interaction occurs between an in-plane magnetic dipole moment of the retinyl chromophore and probably an in-plane electric dipole moment of a nearby aromatic amino acid(s), and that although the membrane is anisotropic with respect to coupling between electric and magnetic moments of the aromatic amino acids, the transition dipole moments of the aromatic amino acids are not preferentially oriented in either direction.     

Effects of Ultraviolet-B Radiation Stress on the Abscisic Acid Status of Rumex patientia Leaves

To determine if increased abscisic acid (ABA) levels are associated with ultraviolet-B (UV-B) radiation (288–320 nm) stress, Rumex patientia L. plants were exposed to visible light with high or low (control) UV-B irradiance and then assayed for ABA by gas chromatography. Though leaf growth was inhibited by the UV-B irradiation, no differences in levels of free ABA were found between leaves from the two treatments after 1, 3 or 5 days of exposure. Unlike the situation in most environmental stresses, increased ABA levels are not associated with UV-B radiation stress. Further, photolysis or isomerization are not likely explanations for the absence of ABA increase. Calculations indicate that if ABA in the leaf were fully exposed to solar UV-B radiation only about 6.7% would be photolyzed by existing daily radiation at 40°N latitude, and 11.3% by radiation resulting from a 40% decrease in atmospheric ozone. The quantum yield for isomerization of cistrans-ABA by radiation in the 288–312 nm waveband was determined. Existing daily solar UV-B irradiation is capable of isomerizing ABA in solution to an equilibrium mixture of 50% cisjrans and 50%irans, trans-ABA., However, because of radiation attenuation in the leaf, it is estimated that only a small fraction of the cistrans-A.BA. would be isomerized.     

A new filter that accurately mimics the solar UV-B spectrum using standard UV lamps: the photochemical properties, stabilization and use of the urate anion liquid filter

The physiological effects unique to solar ultraviolet (UV)-B exposure (280-315 nm) are difficult to accurately replicate in the laboratory. This study evaluates the effectiveness of the sodium urate anion in a liquid filter that yields a spectrum nearly indistinguishable from the solar UV-B spectrum while filtering the emissions of widely used UV-B lamps. The photochemical properties and stability of this filter are examined and weighed against a typical spectrum of ground-level solar UV-B radiation. To test the effectiveness of this filter, light-saturated photosynthetic oxygen evolution rates were measured following exposure to UV-B filtered either by this urate filter or the widely used cellulose acetate (CA) filter. The ubiquitous marine Chlorophyte alga Dunaliella tertiolecta was tested under identical UV-B flux densities coupled with ecologically realistic fluxes of UV-A and visible radiation for 6 and 12 h exposures. These results indicate that the urate-filtered UV-B radiation yields minor photosynthetic inhibition when compared with exposures lacking in UV-B. This is in agreement with published experiments using solar radiation. In sharp contrast, radiation filtered by CA filters produced large inhibition of photosynthesis.     

Photoresponsive polypeptides: Photochromism and conformation of poly (L-glutamic acid) containing spiropyran units

Spirobenzopyran units were bound to the side chains of poly (L -glutamic acid) and partially methylated poly(L -glutamate)s. The modified polymers were found to exhibit “reverse photochromism” in hexafluoro-2-propanol (HFP), so the samples kept in the dark were characterized by an intense absorption band in the visible range of the spectrum, which was completely erased upon exposure to sunlight or irradiation at 500–550 nm. The CD spectra showed that the macromolecules adopted a random coil conformation in the dark, whereas the bleached solutions after exposure to light displayed the typical CD pattern of the α-helix. The back reaction in the dark was accompanied by the progressive decrease of the helix content and recovery of the original disordered conformation. The photoinduced conformational changes resulted in large and reversible viscosity variations. When spiropyran side chains were converted to “spiropyran salts” of trifluoroacetic acid, the system was still photochromic, but the macromolecules were disordered both in the dark and light conditions. However, when appropriate amounts of methanol were added as a cosolvent to the HFP solutions, the system responded to light, giving reversible variations of the α-helix content. Irradiation at appropriate solvent compositions allowed modulation of the extent of the photoresponse. © 1993 John Wiley & Sons, Inc.     

Measurement of leaf epidermal transmittance of UV radiation by chlorophyll fluorescence

In higher plants one of the important functions of the leaf epidermis is the effective screening of ultraviolet-B (280–320 nm, UV-B) radiation, due mostly to phenolic compounds. The assessment of the contribution of this function is necessary for an evaluation of the impact of increasing UV-B radiation. A method is proposed to estimate epidermal transmittance on the basis of chlorophyll fluorescence measurements. Fluorescence of chlorophyll induced by UV-A (320–400 nm, measuring beam centered at 366 nm, half band width 32 nm) or UV-B (measuring beam centered at 314 nm, half band width 18 nm) is compared to that induced by a blue-green measuring light (475 nm, half band width 140 nm). It is shown that the ratios of UV-and blue-green (BG)-induced fluorescence, F(UV-A)/F(BG) and F(UV-B)/F(BG), are relatively constant among leaf samples of various species ( Vicia faba, Spinacia oleracea, Rumex scutatus ) from which the epidermis was removed. In epidermis-free leaves no significant differences were found between adaxial and abaxial leaf sides, suggesting that leaf structure has negligible influence on the F(UV)/F(BG) ratios. On the other hand, fluorescence excitation ratios varied over a vast range when intact leaves from different species and habitats were investigated. Ratios were low in sun leaves and relatively high in shade- and greenhouse-grown leaves. By relating these results to those obtained with epidermis-free leaves, epidermal transmittances for UV-B radiation could be estimated, with values ranging between 1 and 45%. The data demonstrate a large adaptability of epidermal UV-A and UV-B transmittance in higher plants. The proposed method may prove a versatile and relatively simple tool for investigating epidermal UV transmittance complementing established methods.     

Heat stress induces an inhibition of excitation energy transfer from phycobilisomes to photosystem II but not to photosystem I in a cyanobacterium Spirulina platensis.

The effects of high temperature (30-52.5 degrees C) on excitation energy transfer from phycobilisomes (PBS) to photosystem I (PSI) and photosystem II (PSII) in a cyanobacterium Spirulina platensis grown at 30 degrees C were studied by measuring 77 K chlorophyll (Chl) fluorescence emission spectra. Heat stress had a significant effect on 77 K Chl fluorescence emission spectra excited either at 436 or 580 nm. In order to reveal what parts of the photosynthetic apparatus were responsible for the changes in the related Chl fluorescence emission peaks, we fitted the emission spectra by Gaussian components according to the assignments of emission bands to different components of the photosynthetic apparatus. The 643 and 664 nm emissions originate from C-phycocyanin (CPC) and allophycocyanin (APC), respectively. The 685 and 695 nm emissions originate mainly from the core antenna complexes of PSII, CP43 and CP47, respectively. The 725 and 751 nm band is most effectively produced by PSI. There was no significant change in F725 and F751 during heat stress, suggesting that heat stress had no effects on excitation energy transfer from PBS to PSI. On the other hand, heat stress induced an increase in the ratio of Chl fluorescence yield of PBS to PSII, indicating that heat stress inhibits excitation energy transfer from PBS to PSII. However, this inhibition was not associated with an inhibition of excitation energy transfer from CPC to APC since no significant changes in F643 occurred at high temperatures. A dramatic enhancement of F664 occurring at 52.5 degrees C indicates that excitation energy transfer from APC to the PSII core complexes is suppressed at this temperature, possibly due to the structural changes within the PBS core but not to a detachment of PBS from PSII, resulting in an inhibition of excitation energy transfer from APC to PSII core complexes (CP47 + CP43). A decrease in F685 and F695 in heat-stressed cells with excitation at 436 nm seems to suggest that heat stress did not inhibit excitation energy transfer from the Chl a binding proteins CP47 and CP43 to the PSII reaction center and the decreased Chl fluorescence yields from CP43 and CP47 could be explained by the inhibition of the energy transfer from APC to PSII core complexes (CP47 + CP43).     

Comparison of helium-neon and dye lasers for the excitation of allophycocyanin

Allophycocyanin (APC) has a broad absorption spectrum permitting several different lasers to be used to excite this dye in a flow cytometer. A comparison was made between a dye laser and a helium-neon (HeNe) laser for the excitation of APC as an immunofluorescent chromophore. The ratio of fluorescence of stained to unstained lymphocytes (signal to background) was used to assess differences in sensitivity. In determining the best wavelength for operating the dye laser, it was found that there was little difference in the ability to separate the positive-labelled cells from the unstained cells using 600 nm or 633 nm light for excitation of APC. A study of the effect of laser power on the signal to background identified a nonlinear relationship. It was found that the sensitivity obtained with 47 mW of 633 nm light from a HeNe laser was near the maximum attainable. This sensitivity was comparable to that obtained using phycoerythrin as an immunofluorescence chromophore. APC had the added advantage of being applicable to the study of highly autofluorescent cells. Exciting this chromophore using red light dramatically decreased the autofluorescence observed even on alveolar macrophages.     

Damage and recovery from UV-B exposure in conidia of the entomopathogens Verticillium lecanii and Aphanocladium album

We evaluated the effects of exposure to doses supplied at an environmentally realistic intensity of UV-B radiation (800 mW m(-2) weighted irradiance) on the culturability and germination of selected strains of the entomopathogenic Hyphomycetes Verticillium lecanii and Aphanocladium album. Increased UV-B exposure decreased relative percent culturability for all strains. Four hours of exposure to UV-B were sufficient to reduce the culturability close to zero. The LT(50) (50% lethal time) ranged from 120 ± 5 min for the V. lecanii strain ARSEF 6430 to 86 ± 14 min for the A. album strain ARSEF 6433. A strong delay in the germination of surviving conidia was observed. To determine the occurrence of photoreactivation in these two genera, we evaluated the effect of exposure to visible light after exposure to UV-B radiation. There was no significant difference in relative culturability between conidia exposed to visible light after UV-B exposure compared to those incubated in the dark after UV-B exposure. This indicates that photoreactivation, if it occurs, must have limited importance in the repair of the damage induced by UV-B radiation in these two genera.