Structure and function of muscle myosin

We have studied the primary structures of myosins from chicken muscles in order to clarify the relationship between structure and function of muscle myosin. The primary structures of the various kinds of light chains from chicken muscle myosins have been determined. We also report the primary structure of the 23K fragment of subfragment-1 (S-1) component from the heavy chain of chicken fast skeletal muscle myosin. In addition, antibody was prepared against the 23K fragment. The antibody was found to inhibit the Mg²⁺-ATPase activity and the initial P_i burst of the ATPase in the S-1 component. The antibody suppressed the ATP-induced fluorescence enhancement of S-1, though it did not suppress the binding of ATP to S-1. These results are also discussed.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Kinetic properties of prompt and delayed fluorescence of chlorophyll a with λmax= nm when electron transport is blocked on acceptor side of photosystem II

This work is a theoretical consideration of steady-state kinetics of prompt and delayed fluorescence of chlorophyll a entering into the pigment matrices of photosynthetic units of photosystem II when the electron transport from the primary to secondary acceptor of this system is blocked. It has been shown that in such a system of quantum yields of prompt and delayed fluorescence are complementary. At low intensities of excitation light the quantum yield of delayed fluorescence is several times more than that of prompt fluorescence. With an increase in the light intensity the reverse situation is observed. The literature data given sustain the results obtained. it has also been unambiguously shown what values, when changed, may be responsible for the corresponding changes in prompt and delayed fluorescence yields.     

Photoreconvertible fluorophore systems in rhabdomeres,Semper cells and corneal lenses in the compound eye of the blowfly

1. The primary aim of the experiments described in this article was to localize the origin of the complex fluorescence in the compound eye of flies. The eye tissue was dissected and the fluorescence from cells and cell organelles was recorded by microspectrofluorometry. Using this technique, fluorophore systems were detected in the rhabdomeres, Semper cells and corneal lenses. The fluorophore systems are photoreconvertible by UV and blue light. 2. The fluorophore systems in the rhabdomeres and Semper cells are similar. The intensity of the fluorescence from the microvilli is enhanced up to 29 X by adaptation to UV light. The enhancement is inversely related to the rhodopsin content in the microvilli, indicating that the chromophoric group of the fluorophore is not a vitamin A derivative. 3. The enhancement of the fluorescence by UV light strongly depends on pH, suggesting that the photoreconvertible fluorophore systems in the microvilli and Semper cells are photosensitive redox pigments. These redox systems are probably located in the membranes of the microvilli in the photoreceptors, and in the endoplasmic reticulum of the Semper cells, or they are coupled to filaments in the cytoskeleton of both cell types. 4. Preliminary reaction schemes for the photoreactions based on the recorded excitation and emission spectra and photokinetics were developed. A primary pigment in the microvillous structure, AR, or in organelles in the Semper cells, AS, is converted by UV light into an excited state AR* or AS*, which either relaxes to the primary pigment by photon emission, or converts into an intermediate X, which by proton uptake changes into stable products, BR or BS. Blue illumination converts BR and BS into the excited states BR* and BS*, which either relax by photon emission to BR or BS, or convert into an intermediate Y, which after deprotonation reconverts into the primary pigment AR or AS. 5. Estimation of the molecular density showed that the concentration of the fluorophore in the microvilli presumably is almost equal to maximal rhodopsin concentration. The high density suggests that the fluorophores have a specific function in transduction or adaptation of the visual process.     

Oxygen-quenched chlorophyll a fluorescence and electron transport in barley during greening

Seedlings of barley ( Hordeum vulgare L. cv. Conquest) were dark-grown for 7 days and then transferred to light. The time courses of chlorophyll a fluorescence induction underwent changes during greening periods of from 3 to 48 h. Yields of variable fluorescence during greening correlated with electron transport capacity via photosystem II (PS II) except at the early stage from 3 to 6 h. The discrepancy may result from there being only a small amount of light harvesting complex associated with PSII for 3 to 6 h greening. Oxygen quenching effects were interpreted as indicating the development of the electron transport system and the organization of light harvesting complex associated with PSII. The most intensive O₂ quenching of relative fluorescence is found during the early stage of greening when the ratio of the primary quinone electron acceptor (Q) to chlorophyll is high.     

Estimation of Primary Productivity by Chlorophyll a in vivo Fluorescence in Freshwater Phytoplankton

Primary productivity in marine waters is widely estimated by the measurements of ¹⁴C incorporation, the underwater light climate, and the absorption spectra of phytoplankton. In bio-optical models the quantum efficiency of carbon fixation derived from ¹⁴C incorporation rates, the photosynthetically absorbed radiation derived from the underwater light climate, and the phytoplankton absorption spectra are used to calculate time- and depth-integrated primary productivity. Due to the increased sensitivity of commercially available fluorometers, chlorophyll a in vivo fluorescence became a new tool to assess the photosynthetic activity of phytoplankton. Since fluorescence data yield only relative photosynthetic electron transport rates, a direct conversion into absolute carbon fixation rates is not possible. Here, we report a procedure how this problem can be adressed in freshwater phytoplankton. We adapted a marine bio-optical model to the freshwater situation and tested if this model yields realistic results when applied to a hypertrophic freshwater reservoir. Comparison of primary productivity derived from ¹⁴C incorporation to primary productivity derived from Chl a fluorescence showed that the conversion of fluorescence data into carbon fixation rates is still an unsolved problem. Absolute electron transport rates calculated from fluorescence data tend to overestimate primary production. We propose that the observed differences are caused mainly by neglecting the package effect of pigments in phytoplankton cells and by non-carbon related electron flow (e.g., nitrogen fixation). On the other hand, the ¹⁴C incorporation rates can be artificially influenced by "bottle effects", especially near the water surface, where photoinhibition, photorespiration, and Mehler reaction can play a major role.     

The influence of the electric diffusion potential on delayed fluorescence light curves of chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea

The potassium salt-induced transient increase of delayed fluorescence yield was studied in pea chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea.A simple kinetic model is proposed to account for the actinic light intensity dependence of the delayed fluorescence enhancement by the transmembrane diffusion potential induced by sudden salt addition. The electric field dependence of the rate constants for the recombination of primary separated charges with and without subsequent electronic excitation of reaction center chlorophyll was obtained.From the value of enhancement of delayed fluorescence by salt concentration gradients at saturating actinic light intensity, it is concluded that the distance, normal to thylakoid membrane surface, between the primary acceptor and the donor of Photosystem II is smaller than the membrane thickness.     

Standardization of the post-irradiation method to eliminate primary fluorescence in cytofluorometry

Summary Thorough irradiation of specimen with the strong excitation light after fluorescent Feulgen staining destroys the primary fluorescence in the background with stabilization of specific fluorescence of pararosaniline (post-irradiation method).An apparatus to perform effective post-irradiation was developed and irradiation condition for DNA cytofluorometry on a pararosaniline Feulgen stained smear was standardized. After 10 h irradiation on this condition, the primary fluorescence in the background is almost completely cleared away. Proportionality between amount of fluorescence and DNA content is perfectly preserved and standard deviation in each class of ploidy becomes to be less than 5% of the mean DNA value.The standardized post-irradiation enables us to obtain reproducibly the same values of fluorescence on Feulgen stained nuclei of the same cell population in different smears even if the staining conditions in Schiff's solution might be different to some extent.Partly supported by Alexander von Humboldt-Stiftung     

The currently accepted model of primary energy conversion in the plant photosystems must be substantially modernized

According to the current model of primary events in plants, electronic excitations generated in antenna chlorophylls (Chl) by the light rapidly migrate within vast Chl ensembles, reach the reaction centres (RCs) and initiate the primary photoreactions of electron transfer from the RC special pairs (P700 and P680 in plant photosystems). A minor portion of electronic excitations is lost en route, in particular, via fluorescence of Chl a. A number of fluorescence parameters in vivo had been reliably established in many independent studies. Based on these parameters, the author calculated a dimensionless value, the ratio of fluorescence yields emitted by PS-2 and PS-1 Chl a ensembles. The ratio proved to differ 5-10 times from that obtained in experiments. Such a divergence seems to indicate an internal discrepancy in the currently used model. The author proposes a substantial modernization of the model by introducing a new subpicosecond state for RCs, which precedes the primary reaction of electron transfer from the RC special pairs.     

Standardization of the post-irradiation method to eliminate primary fluorescence in cytofluorometry.

Thorough irradiation of specimen with the strong excitation light after fluorescent Feulgen staining destroys the primary fluorescence in the background with stabilization of specific fluorescence of pararosaniline (post-irradiation method). An apparatus to perform effective post-irradiation was developed and irradiation condition for DNA cytofluorometry on a pararosaniline Feulgen stained smear was standardized. After 10 h irradiation on this condition, the primary fluorescente in the background is almost completely cleared away. Proportionality between amount of fluorescence and DNA content is perfectly preserved and standard deviation in each class of ploidy becomes to be less than 5% of the mean DNA value. The standardized post-irradiation enables us to obtain reproducibly the same values of fluorescence on Feulgen stained nuclei of the same cell population in different smears even if the staining conditions in Schiff's solution might be different to some extent.     

Changes of the chlorophyll fluorescence induction kinetics of C3 and CAM plants during day/night cycles

The induction kinetics of the 680 nm chlorophyll fluorescence were measured on attached leaves of Kalanchoë daigremontiana R. Hamet et Perr. (CAM plant), Sedum telephium L. and Sedum spectabile Bor. (C₃ plant in spring, CAM plant in summer) and Raphanus sativus L. (C₃ plant) at three different times during a 12/12 h day/night cycle. During the fluorescence transient the fluorescence intensity at the O, P and T-level (f_O, f_max, f_st,) was different for the plant species tested; this may be due to their different leaf structure, pigment composition and organization of their photosystems. The kinetics of the fluorescence induction depended on the time of preillumination or dark adaptation during the light/dark cycle but not on the type of primary CO₂ fixation mechanism (C₃ and CAM). For dark adapted leaves measured either at the end of the dark phase or after dark adaptation of plants taken from the light phase a higher P-level fluorescence, a higher variable fluorescence (P-O) and a larger complementary area were found than for leaves of plants taken directly from the light phase. This indicates the presence of largely oxidized photosystem 2 acceptor pools during darkness. During the light phase the fluorescence decline after the P-level was faster than during the dark phase; from this we conclude that the light adaptation of the photosynthetic apparatus (state 1state 2 transition, pH) during the induction period proceeded faster in plants taken from the light phase than in plants taken from the dark phase.Abbreviations C₃ plant plant with primary CO₂ fixation on ribulose-1,5-bis-phosphate (Calvin-Benson cycle) - CAM Crassulacean Acid Metabolism     

Changes of the chlorophyll fluorescence induction kinetics of C3 and CAM plants during day/night cycles

The induction kinetics of the 680 nm chlorophyll fluorescence were measured on attached leaves of Kalanchoë daigremontiana R. Hamet et Perr. (CAM plant), Sedum telephium L. and Sedum spectabile Bor. (C₃ plant in spring, CAM plant in summer) and Raphanus sativus L. (C₃ plant) at three different times during a 12/12h day/night cycle. During the fluorescence transient the fluorescence intensity at the O, P and T-level (f_O, f_max, f_st,) was different for the plant species tested; this may be due to their different leaf structure, pigment composition and organization of their photosystems. The kinetics of the fluorescence induction depended on the time of preillumination or dark adaptation during the light/dark cycle but not on the type of primary CO₂ fixation mechanism (C₃ and CAM). For dark adapted leaves measured either at the end of the dark phase or after dark adaptation of plants taken from the light phase a higher P-level fluorescence, a higher variable fluorescence (P-O) and a larger complementary area were found than for leaves of plants taken directly from the light phase. This indicates the presence of largely oxidized photosystem 2 acceptor pools during darkness. During the light phase the fluorescence decline after the P-level was faster than during the dark phase; from this we conclude that the light adaptation of the photosynthetic apparatus (state 1→ state 2 transition, Δ pH) during the induction period proceeded faster in plants taken from the light phase than in plants taken from the dark phase.     

Observations on the fluorescence and function of spiders' eyes

Preliminary observations on spiders' eyes showed that certain eyes fluoresce in ultraviolet light and others do not. The response of these eyes to ultraviolet and visible light has been investigated to discover the relationship, if any, of eye fluorescence with eye function.
In the first part of this paper it is shown that of eight spiders from families with widely differing habits, vision and behaviour, five species reacted to light fluctuations and to differences in brightness of the primary colours blue, green and red. Three species did not respond to lightand only two, S. scenicus and E. falcata , indicated a preference for blue light. It was also found that the visual sensitivity of S. secenicus extended into the ultraviolet. The second part of the paper gives the results of examination in ultraviolet light of the eyes of 40 species from 11 families. Spiders with poor sight and a preference for shade generally showed a strong fluorescence of all eyes. The anterior median and lateral eyes of those species with good sight fluoresced only weakly or not at all, whereas the posterior median and lateral eyes ofthese spiders fluoresced brightly.
Freshly cut frozen sections of the eyes of two selected species, S. scenicus with good sight and C. similis with poor sight, were examined with the fluorescence, phase and polarizing microscopes. The localization of the fluorescence in these eyes is described and a fluorescent substance, common to all the spiders, was found in the lens of the eyes of most species examined.Additional information on the structure of the cornea and lens was also revealed by phase and polarized light microscopy.
The results suggest that spiders' eyes respond to light in different ways and the fluorescent substance present in the lens of the eyes is related to eye function.     

Fluorescence of Dyes in Solutions with High Absorbance. Inner Filter Effect Correction

Fluorescence is a proven tool in all fields of knowledge, including biology and medicine. A significant obstacle in its use is the nonlinearity of the dependence of the fluorescence intensity on fluorophore concentration that is caused by the so-called primary inner filter effect. The existing methods for correcting the fluorescence intensity are hard to implement in practice; thus, it is generally considered best to use dilute solutions. We showed that correction must be performed always. Furthermore, high-concentration solutions (high absorbance) are inherent condition in studying of the photophysical properties of fluorescent dyes and the functionally significant interactions of biological macromolecules. We proposed an easy to use method to correct the experimentally recorded total fluorescence intensity and showed that informative component of fluorescence intensity numerically equals to the product of the absorbance and the fluorescence quantum yield of the object. It is shown that if dye molecules do not interact with each other and there is no reabsorption (as for NATA) and spectrofluorimeter provides the proportionality of the detected fluorescence intensity to the part of the absorbed light (that is possible for spectrofluorimeter with horizontal slits) then the dependence of experimentally detected total fluorescence intensity of the dye on its absorbance coincides with the calculated dependence and the correction factor for eliminating the primary inner filter effect can be calculated on the basis of solution absorbance. It was experimentally shown for NATA fluorescence in the wide range of absorbance (at least up to 60). For ATTO-425, which fluorescence and absorption spectra overlap, the elimination of the primary and secondary filter effects and additional spectral analysis allow to conclude that the most probable reason of the deviation of experimentally detected fluorescence intensity dependence on solution absorbance from the calculated dependence is the dye molecules self-quenching, which accompanies resonance radiationless excitation energy transfer.     

Photosystem II Photosynthetic Unit Sizes from Fluorescence Induction in Leaves : CORRELATION TO PHOTOSYNTHETIC CAPACITY

The use of fluorescence induction measurements in leaves infiltrated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea has been evaluated as a routine method for estimation of the concentration of the reaction centers of photosystem II relative to total chlorophyll in a wide variety of plant species. The procedure is based on a simple theory that takes into account the attenuation of light in passing through the leaf and the linear dependence of the fluorescence induction time from different parts of the leaf on the inverse of the local light intensity. A formula to calculate the reaction center concentration of photosystem II was obtained. The effect of the light attenuation is accounted for by a correction factor which could become practically insignificant by an optimal choice of the excitation and emission wavelengths and the geometry of the photodetector with respect to the sample. Estimation of quantum yields for primary photochemistry and influence of light scattering were considered. The results demonstrate the effect of the above factors under various circumstances and are in agreement, to a first approximation, with the theory.     

Long-range interactions of <Emphasis Type="Italic">Chara</Emphasis> chloroplasts are sensitive to plasma-membrane H<Superscript>+</Superscript> flows and comprise separate photo- and dark-operated pathways

Local illumination of the characean internode with a 30-s pulse of white light was found to induce the delayed transient increase of modulated chlorophyll fluorescence in shaded cell parts, provided the analyzed region is located downstream in the cytoplasmic flow at millimeter distances from the light spot. The fluorescence response to photostimulation of a remote cell region indicates that the metabolites produced by source chloroplasts in an illuminated region are carried downstream with the cytoplasmic flow, thus ensuring long-distance communications between anchored plastids in giant internodal cells. The properties of individual stages of metabolite signaling are not yet well known. We show here that the export of assimilates and/or reducing equivalents from the source chloroplasts into the flowing cytoplasm is largely insensitive to the direction of plasma-membrane H⁺ flows, whereas the events in sink regions where these metabolites are delivered to the acceptor chloroplasts under dim light are controlled by H⁺ fluxes across the plasma membrane. The fluorescence response to local illumination of remote cell regions was best pronounced under weak background light and was also observed in a modified form within 1–2 min after the transfer of cell to darkness. The fluorescence transients in darkened cells were suppressed by antimycin A, an inhibitor of electron transfer from ferredoxin to plastoquinone, whereas the fluorescence response under background light was insensitive to this inhibitor. We conclude that the accumulation of reduced metabolites in the stroma leads to the reduction of photosystem II primary quinone acceptor (Q_A) via two separate (photochemical and non-photochemical) pathways.     

Chlorophyll a fluorescence in phytoplankton: relationship to photosynthesis and biomass

The theoretical and applied aspects of in vivo chlorophyll fluorescenceare reviewed for aquatic biologists who use fluorescence inestimating standing stocks and photosynthetic activity. Themajor advantage of using fluorescence is that the measurementis easy to make. However, despite some sound theoretical modelsdescribing variable fluorescence there are many environmentalfactors influencing fluorescence about which little is known.Much more basic research on fluorescence-photosynthesis relationshipsneeds to be done before fluorescence per se can replace current¹⁴C or O₂ methods for measuring primary productivity. ^*This paper is the result of a study made at the Group for AquaticPrimary Productivity (GAP), Second International Workshop heldat the National Oceanographic Institute, Haifa, Israel in April–May1984.     

盐胁迫对不同抗盐性小麦叶片荧光诱导动力学的影响

以不同抗盐性小麦为材料,研究了NaCl胁迫对叶片叶绿素。荧光诱导动力学的影响。指出150 mmol/L NaCl使小麦幼苗生长降低,叶绿素含量下降,同时使光系统Ⅱ的潜在光化学活性和原初光能转化率降低,使光反应受到抑制。     

A liquid crystal pixel array for signal discrimination in array biosensors

A new optical design uses a liquid crystal pixel array (LCPA) to discriminate multiple fluorescence signals on a two-dimensional biosensor array. The LCPA can selectively control the transmission of fluorescence generated from multiple biosensing elements on a planar waveguide. This device sequentially acquires the fluorescence data from the substrate by making multiple individual measurements of the sensing elements on the waveguide. The biosensing elements are patterned according to the pixel layout of the LCPA and optically aligned so that each electronically driven pixel can either transmit or filter out the fluorescence signal as specified by the user. The primary advantage of this system is that a single detection channel (i.e. photomultiplier tube (PMT)) can be used to measure multiple fluorescence signals from a two-dimensional substrate while the LCPA provides for spatial resolution. We evaluate the performance of the LCPA by testing the optical homogeneity of the liquid crystal pixels and linear dynamic range for transmitting light. The LCPA is also used with well-developed biosensing chemistry modified for this optical format.     

用体内叶绿素a荧光诱导动力学鉴定番茄的抗冷性

研究了冷害温度对具有不同抗冷性品种的番茄叶片的体内叶绿素a荧光诱导动力曲线的影响。实验结果指出,在低温处理(8℃,5℃,2℃下,暗中24小时)后,番茄叶片的体内叶绿素a荧光诱导动力学曲线有了明显的改变,Fv／Fo值、Rfd值降低了,光系统II原初光能转换效率和潜在的光合活力均受到抑制。我们在苗期和开花期得到的实验结果均表明,在番茄叶片的叶绿素a荧光诱导动力学曲线和这些荧光参数改变的程度与该品种的已知抗冷性之间呈现较好的相关性。我们认为,体内叶绿素a荧光诱导动力学方法是鉴定番茄抗冷性的一个快速、灵敏和可靠的方法,并可用于其他绿色植物的抗冷性鉴定中。     

Combinatorial fluorescence energy transfer molecular beacons for probing nucleic acid sequences.

We report the design, synthesis, and characterization of molecular beacons (MB) consisting of three distinct fluorophores, 6-carboxyfluorescein (Fam), N,N,N',N'-tetramethyl-6-carboxyrhodamine (Tam), and Cyanine-5 (Cy5). The primary light absorber/energy donor (Fam) is located on one terminus of the MB, whereas the primary energy acceptor/secondary donor (Tam) and secondary acceptor (Cy5) are located at the other terminus of the MB. In the absence of target DNA or RNA, the MB exists in the stem-closed form. Excitation of Fam initiates an energy transfer cascade from Fam to Tam and further to Cy5 generating unique fluorescence signatures defined as the ratio of the emission from each of the three fluorophores. This energy transfer cascade was investigated in detail by steady-state and time-resolved fluorescence spectroscopy, as well as fluorescence depolarization studies. In the presence of the complementary target DNA, the MB opened efficiently and hybridized with the target separating Fam and Tam by a large distance, so that energy transfer from Fam to Tam was blocked in the stem-open form. This opening of the MB generates a "bar code" fluorescence signature, which is different from the signature of the stem-closed MB. The fluorescence signature of this combinatorial fluorescence energy transfer MB can be tuned by variation of the spacer length between the individual fluorophores.