Non-local interactions between light induced processes inCalliphora photoreceptors

Summary The prolonged depolarizing afterpotential (PDA) is a phenomenon which is tightly linked to visual pigment conversion. In order to determine whether processes underlying PDA induction and depression can spread in space, the PDA was recorded intracellularly in white-eyedCalliphora R1-6 photoreceptors and used to examine interactions between processes induced by activating statistically different photopigment molecules (Figs. 3–6). It was found that a PDA induced by converting some fraction of rhodopsin (R) molecules forward into the metarhodopsin (M) state can be completely depressed by equal or smaller amounts of pigment conversion, backward from metarhodopsin to rhodopsin even when largely different sets of pigment molecules were shifted in the respective directions, in agreement with previous experiments conducted on the barnacle. The characteristics of the afterpotentials obtained following the cessation of strong blue and green light stimuli which did not cause a net pigment conversion was examined (Figs. 7, 8). It was found that these afterpotentials, obtained when nonet R to M conversion took place, could not be depressed by an opposite net large M to R pigment conversion. Accordingly we propose to restrict the term PDA to an afterpotential which can be depressed by a net M to R pigment conversion. It is concluded: (a) that some processes underlying PDA induction and depression inCalliphora must interact at a distance which extends at least to the nearest neighboring pigment molecule, and (b) that inCalliphora photoreceptors net pigment conversion is required in order to induce and depress a PDA.Abbreviations R rhodopsin - M metarhodopsin - R to M rhodopsin to metarhodopsin pigment conversion - M to R metarhodopsin to rhodopsin pigment conversion - PDA prolonged depolarizing afterpotential - ERG electroretinogram - M potential metarhodopsin potential - ERP early receptor potential     

Nanopore sensor for fast label-free detection of short double-stranded DNAs

Functionalizing surface enhanced the molecular sensing ability of a fabricated nanopore by increasing the translocation duration time for a short double-stranded DNA. The surface of nanopore was derivatized with γ-aminopropyltriethoxysilane and the positively charged surface attracted DNA molecules when they were in the vicinity of nanopore. The translocation duration time of DNA increased due to the strong electrostatic interaction and it enabled us to detect a short double-stranded DNA (<1 kbp) that is under the size limit of a conventional solid state nanopore sensor. Both 539 and 910 bp double-stranded DNAs were analyzed with the surface functionalized nanopore and their translocation kinetics are presented in this work. The new feature of the surface modified nanopore that can detect short double-stranded DNA molecules could readily be applied for a rapid label-free diagnostic analysis in a Lab-On-a-Chip type DNA sensor.     

A mechanistic model for the light response of photosynthetic electron transport rate based on light harvesting properties of photosynthetic pigment molecules

Models describing the light response of photosynthetic electron transport rate (ETR) are routinely used to determine how light absorption influences energy, reducing power and yields of primary productivity; however, no single model is currently able to provide insight into the fundamental processes that implicitly govern the variability of light absorption. Here we present development and application of a new mechanistic model of ETR for photosystem II based on the light harvesting (absorption and transfer to the core ‘reaction centres’) characteristics of photosynthetic pigment molecules. Within this model a series of equations are used to describe novel biophysical and biochemical characteristics of photosynthetic pigment molecules and in turn light harvesting; specifically, the eigen-absorption cross-section and the minimum average lifetime of photosynthetic pigment molecules in the excited state, which describe the ability of light absorption of photosynthetic pigment molecules and retention time of excitons in the excited state but are difficult to be measured directly. We applied this model to a series of previously collected fluorescence data and demonstrated that our model described well the light response curves of ETR, regardless of whether dynamic down-regulation of PSII occurs, for a range of photosynthetic organisms (Abies alba, Picea abies, Pinus mugo and Emiliania huxleyi). Inherent estimated parameters (e.g. maximum ETR and the saturation irradiance) by our model are in very close agreement with the measured data. Overall, our mechanistic model potentially provides novel insights into the regulation of ETR by light harvesting properties as well as dynamical down-regulation of PSII.     

Effects of energy deprivation on the fly pupil mechanism: evidence for a rigor state

The energy dependence of the pupil pigment-migrations in the fly Musca domestica was studied in live animals, using optical techniques and nitrogen-gas induced anoxia. The results obtained can be summarized in 3 points:

1. Energy deficiency can make the pupil mechanism stop in any state, extreme or intermediate.
2. Anoxia induced during intermittent stimulation makes the pupil stop in the closed state (aggregated pigment granules).
3. During long-term anoxia the pupil very slowly opens (dispersal of pigment granules), irrespective of ambient intensity.

The slow anoxic opening (point 3) is more than 1000 times slower than that predicted for free diffusion of pigment granules in water. Assuming realistic values of cytoplasm viscosity, this implies that anoxia causes the pigment granules to attach to rigid structures in the cells, in analogy with the rigor state in anoxic muscles. The rigor phenomenon in the pupil mechanism prevents experimental discrimination between active and passive processes of pigment migration. Normal pupil opening has a time course which agrees reasonably with a passive diffusion process, but it is argued that an active transportation of granules away from the rhabdom is more likely in the dark adapted eye.     

Phase transition kinetics of phosphatidic acid bilayers A stopped-flow study of the electrostatically induced transition

The kinetics of the electrostatically induced phase transition of dimyristoyl phosphatidic acid bilayers was followed using the stopped-flow technique. The phase transition was triggered by a fast change in the pH or the magnesium ion concentration and followed by recording the time dependence of the absorbance. When the phase transition was induced by a pH jump the time course of the absorbance could be described by two exponentials, their time constants displaying the for cooperative processes characteristic maximum at the transition midpoint. The time constants are in the 10 and 100 ms range for the H⁺ triggered transition from the fluid to the ordered state. A third slower process shows no appreciable temperature dependence and is probably caused by vesicle aggregation. For the OH^--induced transition fron the ordered to the fluid state the time constants are in the 100 and 1000 ms range. The fluid-ordered transition could also be triggered by addition of magnesium ions. Of the several observed processes only the fastest in the 10–100 ms time range could definitely be assigned to the fluid-ordered transition while the others are due to aggregation phenomena. The experimental data were compared with results obtained from pressure jump experiments and could be interpreted on the basis of theories for non-equilibrium relaxation.     

Photothermal Spectra of Thylakoids Isolated from Cucumber Cotyledons at Various Stages of Greening

The character of interaction between carotenoids (Cars) and chlorophylls (Chls) in thylakoids isolated from cucumber cotyledons at three stages of greening (3, 6, and 24 h of irradiation with 120 µmol m^–2 s^–1) was studied. The shapes of the steady state photoacoustic spectra were changed with the change in time of greening and with the frequency of radiation modulation. The shapes show that changes not only in the contents of various pigments but also in pigment interactions with surrounding occur and that processes of thermal deactivation characterised by different kinetics take place. Slow processes of thermal deactivation are in most cases due to deactivation of triplet states. Long living triplet states are very often engaged in photochemical reactions that can destroy the tissue. Analysis of the time-resolved photothermal spectra shows that at later stage of greening, the chlorophyll (Chl) molecules are better shielded against photo-destruction because Cars more efficiently quench their triplet states. The yield of formation of the pigment triplet states measured by the time resolved photothermal method, always at the same energy absorbed by pigment mixture, declined during sample greening. The decay time of the slow component of pigment thermal deactivation, due predominantly to deactivation of the triplet state of Chl, decreases with the increase of time of greening from 6.2 µs for the 3-h sample to 1.5 µs for the 24 h sample. The energy taken by Cars from Chls is dissipated into heat, therefore the steady state and quick thermal deactivation values increased during the greening process. The Cars/Chls ratio in the thylakoids decreased during greening approximately 2 fold. Hence at a later phase of greening the Cars can quench the triplet states of Chls more efficiently than at an earlier phase of greening.     

LIPOFUSCIN (AGING) PIGMENT GRANULES OF THE NEWBORN HUMAN LIVER

We have observed pigmented cytoplasmic granules, with the characteristic staining properties of lipofuscin (ceroid, "wear-and-tear") pigment, in newborn human liver. The pigment is found at the periphery of the lobule in hepatocytes and some bile ductular cells. It is acid-fast, PAS-positive after diastase digestion, slightly argyophilic and sudanophilic, and markedly Schmorl's- and peroxidase positive in paraffin sections. Difficult to see in sections stained with hematoxylin and eosin, the pigment can be detected in unstained sections. The granules also resemble lipofuscin found in adult tissues, in their ultra-structural and enzymatic properties. They are polymorphic, contain granular material of moderate and high electron opacity, and are delimited by a single membrane. Acid phosphatase and β-glucuronidase activities are visualized in the newborn granules, identifying them as lysosomes. The granules also contain copper and, to a much lesser extent, iron. The accumulation of lipofuscin pigment in lysosomes in many tissues correlates well with aging, and this process has been interpreted as a reflection of cellular degeneration or wear-and-tear. However, the presence of lipofuscin granules as a constant component of neonatal liver suggests that they are not a measure of cellular senescence.     

Intermolecular Transmembrane Domain Interactions Activate Integrin αIIbβ3

Integrins are the major cell adhesion molecules responsible for cell attachment to the extracellular matrix. The strength of integrin-mediated adhesion is controlled by the affinity of individual integrins (integrin activation) as well as by the number of integrins involved in such adhesion. The positive correlation between integrin activation and integrin clustering had been suggested previously, but several trials to induce integrin clustering by dimerization of the transmembrane domain or tail region of integrin α subunits failed to demonstrate any change in integrin activation. Here, using platelet integrin αIIbβ3 as a model system, we showed that there is intermolecular lateral interaction between integrins through the transmembrane domains, and this interaction can enhance the affinity state of integrins. In addition, when integrin clustering was induced through heteromeric lateral interactions using bimolecular fluorescence complementation, we could observe a significant increase in the number of active integrin molecules. Because the possibility of intermolecular interaction would be increased by a higher local concentration of integrins, we propose that integrin clustering can shift the equilibrium in favor of integrin activation.     

Dichotomous effect of monocyte Fc receptor interaction on anti-CD3-induced immunoglobulin synthesis

Monoclonal antibodies against the TCR/CD3 complex are capable of activating T cells which in turn may induce immunoglobulin synthesis in B cells under appropriate conditions. Here we present evidence that distinct immune responses, induced by four commonly used TCR/CD3 mAb (Leu4, OKT3, BMA030, BMA031) were related to the mAb interaction with monocyte Fc receptors for IgG. Depending on their isotype and on the technique by which they were crosslinked, TCR/CD3 mAb induced variable IgM and IgG synthesis in PBMC: If the mAb were crosslinked by monocyte IgG-Fc receptors they induced a high Ig production, while crosslinking the same mAb by plastic-bound goat anti-mouse antibodies (panning) failed to do so. Nevertheless, both crosslinking techniques triggered a strong proliferation and IL-2, IL-4, and IFN gamma lymphokine gene expression. The lack of Ig production under panning conditions was due to an additional IgG-Fc receptor interaction with monocytes: (a) If namely mAb F(ab')2 fragments, or mAb isotypes unable to bind to monocyte Fc receptors (IgG2b, IgG1 in nonresponders) were crosslinked by panning, both a good proliferation as well as Ig production ensued; (b) if TCR/CD3 mAb isotypes which could additionally bind to monocyte Fc receptor (IgG2a) were crosslinked, no Ig production occurred; (c) if mAb F(ab')2 fragments were crosslinked with a second anti-T cell antibody of IgG2a isotype, which could bind to monocyte Fc receptors, Ig synthesis was reduced. Interestingly enough, this diminishing effect, due to monocyte Fc receptor interaction, was only observed if CD4-positive cells were proliferating, but not if CD8-positive cells were activated.     

THE FINE STRUCTURE OF LIPOFUSCIN AGE PIGMENT IN THE NERVOUS SYSTEM OF AGED MICE

An examination of the topographic distribution of lipofuscin pigment granules with the light and electron microscope revealed either smaller and randomly "dispersed" or larger and more complex "clustered" pigment configurations in the cytoplasm of neurons in the dorsal ganglia and ventral spinal cord of 24-month old male mice. Qualitative comparisons revealed no major differences in shape, size, complexity, density, orientation, and cytologic distribution of the pigment bodies in motor and sensory neurons. In general, when the pigment granules were quite numerous within the 2 types of cells, they were smaller in size (~lµ), had a dense homogeneous matrix with few bands or lamellae, and were uniformly distributed throughout the cytoplasm. In contrast, when the pigment configurations were less in number, they were usually larger in size (~3µ), had a more complex internal banded structure, and appeared more localized within the cell. Examination of the bands revealed a repeating pattern of ~70 A. The bands appeared to fuse, forming hexagonal arrays of linear densities intersecting at an angle of approximately 120° in some regions of the pigment bodies. Structural similarities suggested that the striated membranous bands may be composed of phospholipids.     

Spatial properties of the prolonged depolarizing afterpotential in barnacle photoreceptors. I. The induction process

In invertebrate photoreceptors, when the light stimulus results in substantial net transfer of the visual pigment from the rhodopsin (R) to the metarhodopsin (M) state, the ordinary late receptor potential (LRP) is followed by a prolonged depolarizing afterpotential (PDA). The dependence of the amplitude of the PDA on the amount of pigment conversion is strongly supralinear, and the PDA duration also depends on this amount. These observations indicate an interaction among the elements of the PDA induction process and also make possible a test of the range of this interaction. The test consists of a comparison of the PDA after localized pigment conversion, obtained by strong spot illumination, to that after weaker diffuse illumination converting a comparable total amount of pigment. The experiment was performed on the barnacle lateral eye. The effective spot size was measured by the early receptor potential (ERP), in seawater saturated with CO2, which considerably reduced the electrical coupling between the photoreceptors. The ERP was also used to determine whether there is diffusion of R molecules into the illuminated spot. The spot illumination induced a PDA with small amplitude and long duration, while no detectable PDA was induced by the diffuse light. This indicates that the range of the PDA interaction is much smaller than the entire cell. In addition, the ERP results showed that there was no detectable diffusion of R molecules into the illuminated spot area over 30 min. This measurement, with a calculated correction for the microvillar geometry of the photoreceptor, enabled us to put an upper limit on the diffusion coefficient of the pigment molecules in the inact, unfixed barnacle photoreceptor of D less than 6 X 10(-9) cm2 s-1.     

Kinetics of rearrangement of solvation sheath of an excited molecule of the fluorescent probe 4"-dimethylaminochalcone

Processes accompanying the quenching of the fluorescent probe 4"-dimethylaminochalcone by hydroxyl groups of the proton-donor solvent 1-butanol have been studied. The kinetics of the deactivation of the excited state of 4"-dimethylaminochalcone has been monitored from the transition absorption spectra at a time resolution of 50 fs and fluorescence decay at a time resolution of 30 ps. The data obtained allow thinking that the next picture occurs in 1-butanol. At first stage, the 4"-dimethylaminochalcone molecule in its ground state forms a hydrogen bond with an alcohol molecule. At the second stage, the absorption of light quantum and corresponding rise of the dipole moment of 4"-dimethylaminochalcone take place, the initially existing hydrogen bond is retained. The third stage consists in the rearrangement of the 4"-dimethylaminochalcone solvation shell formed by alcohol dipole molecules due to an increase of the dipole of moment 4"-dimethylaminochalcone; this rearrangement takes an energy of about 24 kJ/mol, the arrangement time constant is close to 40 ps; the initial hydrogen bond is retained. The fourth stage involves processes that lead to fluorescence quenching; their time constant is about 200 ps. Taking into account that the quenching is a much slower process than the relaxation of the solvation shell, it was supposed that the quenching is not a direct consequence of the solvation shell relaxation or the existence of the hydrogen bond formed prior to excitation. Then the fluorescence quenching of 4"-dimethylaminochalcone can be accomplished through some other processes that are observed in other fluorescent molecules: (a) rearrangement of the initial hydrogen bond from a conformation that cannot quench the fluorescence of 4"-dimethylaminochalcone to a more "effective" conformation, (b) charge transfer between the excited of molecule 4"-dimethylaminochalcone and alcohol, or (c) solvent-induced twist of the 4"-dimethylaminochalcone amino group (its withdrawal from the molecule plane) by the action of the solvent.     

Pigment-pigment interactions and secondary structure of reconstituted algal chlorophyll a/b-binding light-harvesting complexes of Chlorella fusca with different pigment compositions and pigment-protein stoichiometries

Earlier we have shown by in vitro reconstitution experiments that the pigment composition of the chlorophyll alb-binding light-harvesting complex of the green alga Chlorella fusca could be altered in a relatively broad range (Meyer and Wilhelm 1993). In this study we used these reconstituted complexes of different pigment loading to analyze the excitonic interactions between the pigment molecules and the secondary structure by means of circular dichroism spectra in the visible and the far UV spectral regions, respectively. We found that, in contrast to the expectations, the pigment composition and pigment content hardly affected the circular dichroism spectra in the visible spectral region. Reconstituted complexes, independent of their pigment composition, exhibited the most characteristic circular dichroism bands of the native light-harvesting complex, even if one polypeptide bound only 3 chlorophyll a, 3 chlorophyll b and 1–2 xanthophyll molecules. Full restoration of the protein secondary structure, however, could not be achieved. The -helix content depended significantly on the pigment composition as well as on the pigment-protein ratio of the reconstituted complexes. Further binding of pigments resulted in restoration of the minor excitonic circular dichroism bands, the amplitudes of which depended on the pigment content of the reconstituted complexes. These data suggest that in the reconstitution of light-harvesting complexes a central cluster of pigment molecules plays an important role. Further binding of pigments to the peripheral binding sites appeared also to stabilize the protein secondary structure of the reconstituted complexes.Abbreviations CD- circular dichroism - LHC- chlorophyll a/b light-harvesting complex(es) - LHC II- light-harvesting complex(es) of Photosystem II of higher plants - LHCP- light-harvesting Chl a/b-binding protein(s) - PP- polypeptide(s)     

Dual excitatory innervation through the different ganglionic roots in the uropod adductor muscle of a crayfish,Procambarus clarkii

Dual excitatory innervation of the crayfish (Procambarus clarkii) uropod adductor muscle, adductor exopodite (ADX), through different roots of the sixth abdominal ganglion, was confirmed histologically and electrophysiologically. ADX contraction induced by the excitor in the third root (R3 excitor), showed more rapid fatigue than that by the excitor in the second root (R2 excitor), possibly owing to a more rapid depletion of the transmitter substance in the process of neuromuscular transmission. The size and position of the cell bodies of the two excitors were not necessarily similar, whereas the dendritic fields appeared to be almost overlapping.     

Biological and molecular correlates between induced dedifferentiation and spore germination in Dictyostelium.

When developing cultures of Dictyostelium discoideum are disaggregated at any time prior to cell wall formation and challenged to reinitiate development, amoebae will progress through the original sequence of morphogenetic stages, but the second time through they will do so in roughly one-tenth the original time, a process known as 'rapid recapitulation'. However, if disaggregated cells are suspended in nutrient medium, they enter a program of dedifferentiation during which they lose the capacity to rapidly recapitulate after an 80 minute lag period in a process known as 'erasure'. Here we show that cells that have completed the morphogenetic program and emerge from spore coats in the process of germination have also erased. In addition, the germination-specific 270 gene family is expressed during induced dedifferentiation in a unique fashion, and a germination-defective mutant exhibits a dramatic delay in erasure without concomitant defects in the program of gene regulation accompanying induced dedifferentiation. These results suggest for the first time that induced dedifferentiation and spore germination share some common processes in converting cells from a developmental to vegetative state.     

THE VISUAL CELLS AND VISUAL PIGMENT OF THE MUDPUPPY, NECTURUS

Electron microscopy of the visual cells of the mudpuppy Necturus have revealed several new or hitherto neglected features of organization: (a) A system of deeply staining micelles in virtually crystalline array, probably located in the lamellae of the rod outer segments. These particles may contain the visual pigment, porphyropsin. Counts of the micelles, and microspectrophotometric measurements of porphyropsin in the retina and single rods yield the estimate that each lamellar micelle may contain about 50 molecules of porphyropsin. (b) Systems of about 30 cytoplasmic filaments (here called dendrites), continuous with the cytoplasm of the inner segment, and standing like a palisade about the outer segments of the rods and cones. In the rods, one such filament stands in the mouth of each of the approximately 30 deep fissures that carve the outer segment into a radial array of lobules. (c) A system of deeply staining particles in the membranes of the dendrites, and another in the membranes of the pigment epithelial processes. It is suggested that these may have a part in interchanges of material with the outer segments. The ciliary process is found to penetrate more deeply than is commonly supposed into the outer segments of the rods and cones. The edge of each double-membrane disc in rods forms a differentiated rim structure, both around the disc circumference and bordering the fissures. These anatomical arrangements are summarized in Figs. 13 and 14, and the relevant measurements in Table I. The dilution of visual pigment in Necturus rods and cones and a general consideration of their microstructures make it seem unlikely that such typically solid state processes as exciton migration or photoconduction can transport the effects of light far from the site of absorption. Excitation must, therefore, be conveyed to the receptor as a whole by some axial structure. Among axial structures, the plasma membrane is most likely to be the site of nervous excitation. The ciliary process probably plays its main role in the embryogenesis and regeneration of outer segments; and the dendrites and pigment epithelial processes in exchanges of material with the outer segments and perhaps with one another.     

Epigenetic selection: an alternative mechanism of pattern formation

It is commonly assumed that patterned development is specified by pre-patterns or programs, actual development being a necessary consequence of previous conditions. However, the variability of normal development and its regenerative capacities are evidence for additional patterning processes. Predictable mature structures could result from continued "epigenetic selection" of the most appropriate developmental events. This selection would occur from an excess of possibilities that are genetically equivalent. The final balanced state would be specified by the genes, and the developmental system could gravitate towards this state without a detailed program. Selection could result from competition between cells and tissues for limiting developmental signals. The success or continuation of events that could start randomly would depend on feedback relationships with complementary developmental events. Specialized processes could be gradually localized if differentiation itself consumed limiting signals and if this consumption increased as differentiation proceeded. Spatial patterns could be formed if the movement of signals was gradually facilitated along the axes where it were initiated by diffusion. For example, induced facilitated transport could be the basis of an advantage of multicellular centers over scattered cells that have specialized in the same way. If epigenetic selection has a developmental role it requires a revision of common views concerning the cellular traits and the gene functions necessary for patterned development. An example of these traits is that cells should be expected to respond to changes in signal availability, not necessarily to signal concentration at any given time.     

Small molecule screening identifies targetable zebrafish pigmentation pathways

Small molecules complement genetic mutants and can be used to probe pigment cell biology by inhibiting specific proteins or pathways. Here, we present the results of a screen of active compounds for those that affect the processes of melanocyte and iridophore development in zebrafish and investigate the effects of a few of these compounds in further detail. We identified and confirmed 57 compounds that altered pigment cell patterning, number, survival, or differentiation. Additional tissue targets and toxicity of small molecules are also discussed. Given that the majority of cell types, including pigment cells, are conserved between zebrafish and other vertebrates, we present these chemicals as molecular tools to study developmental processes of pigment cells in living animals and emphasize the value of zebrafish as an in vivo system for testing the on- and off-target activities of clinically active drugs.     

Ultrastructural differences as a taxonomic marker: the segmental ocelli of Polyophthalmus pictus and Polyophthalmus qingdaoensis sp.n. (Polychaeta,Opheliidae)

The segmental ocelli (eyes) in specimens of a European and a Chinese Polyophthalmus pictus population have been investigated by transmission electron microscopy. The ocelli are situated in corresponding positions in the same segments and reveal similarities in their general structure. They consist of one photoreceptor cell with microvilli-bearing processes and a pigment cup, the receptor processes project into an extracellular cavity formed by the sensory cell and a few supporting cells, the pigment cup is formed by mesodermal cells, and basiepidermal glial cells and gland cells lie above the sensory cell. However, the ocelli differ in size and number of cells, number and dimensions of cellular elements as well as presence or absence of certain cell types associated with the ocelli. There is only little variation in these characters and there is no overlap, so that they distinctly separate the specimens of the two populations. These differences are in the same range, or even larger, as those observed between the ocelli of other closely related polychaete species. Therefore, the specimens from Qingdao, China, are described as a new species of the Opheliidae, Polyophthalmus qingdaoensis sp.n., although specimens from Qingdao, China, and the Island of Giglio, Italy, are almost inseparable by light microscopy except for a few subtle differences.