CRITICAL ILLUMINATION AND FLICKER FREQUENCY,AS A FUNCTION OF FLASH DURATION: FOR THE SUNFISH

From the relations between critical illumination in a flash (I_m) and the flash frequency (F) for response of the sunfish to visual flicker when the proportion of light time to dark time (t_L/t_D) in a flicker cycle is varied at one temperature (21.5°) the following results are obtained: At values of t_L/t_D between 1/9 and 9/1 the F - log I_m curves are progressively shifted toward higher intensities and lower F_max.. F_max. is a declining rectilinear function of the percentage of the flash cycle time occupied by light. The rod and the cone portions of the flicker curve are not shifted to the same extent. The cone portion and the rod region of the curve are each well described by a probability integral. In terms of F as 100 F/F_max. the standard deviation of the underlying frequency distribution of elemental contributions, summed to produce the effect proportional to F, is independent of t_L/t_D. The magnitude of log I_m at the inflection point (r''), however, increases rectilinearly with the percentage light time in the cycle. The proportionality between I_m and σ_II1 is independent of t_L/t_D. These effects are interpreted as consequences of the fact that the number of elements of excitation available for discrimination of flicker is increased by increasing the dark interval in a flash cycle. Decreasing the dark interval has therefore the same kind of effect as reducing the visual area, and not that produced by decreasing the temperature.     

THEORY AND MEASUREMENT OF VISUAL MECHANISMS : V. FLASH DURATION AND CRITICAL INTENSITY FOR RESPONSE TO FLICKER

The relation between flash duration and mean critical intensity (white light) for threshold recognition of visual flicker, as a function of flash frequency, was investigated by means of measurements at five values of the light-time fraction: 0.10, 0.25, 0.50, 0.75, 0.90, with flash frequencies of the interrupted beam ranging from 2 to 60 per second. A square area, 6.1 x 6.1°, centrally fixated) was viewed monocularly; the discriminometer used provides automatically an artificial pupil 1.8 mm. in diameter. Except for the slight day-to-day fluctuation in the magnitudes of the parameters, the data for the observer used are shown to form an essentially homogeneous group. As for other animals tested, the F - log I_m curve is enlarged and moved toward lower flash intensities as the light-time fraction is decreased. The high intensity segments of the duplex curves are fitted by normal probability integrals for which F _max. and the abscissa of inflection are rectilinear functions of t_L(t_L + t_D), with opposite slopes. The third parameter, (σ''_{log I}, is invariant. The low intensity segments are composites, their shapes determined by the summation of the lower part of the high intensity curve with an overlapping low intensity population of effects. Both the rising and the declining branches of this latter assemblage suffer competitive partial suppression by the effects in the high intensity population. The detailed analysis shows that these results are consistent with the theory of the central, rather than peripheral, location of the dynamically recognizable elements in the determination of flicker.     

THEORY AND MEASUREMENT OF VISUAL MECHANISMS : III. ΔI AS A FUNCTION OF AREA,INTENSITY, AND WAVE-LENGTH,FOR MONOCULAR AND BINOCULAR STIMULATION

Measurements of ΔI as a function of retinal area illuminated have been obtained at various levels of standard intensity I ₁, using "white" light and light of three modal wave-lengths (λ465, 525, 680), for monocular stimulation and for simultaneous excitation of the two eyes ("binocular"), using several methods of varying (rectangular) area and retinal location, with control of exposure time. For data homogeneous with respect to method of presentation, log ΔI_m = -Z log A + C, where ΔI = Ĩ ₂ – I ₁, A is area illuminated, and C is a terminal constant (= log ΔI_m for A = 1 unit) depending on the units in which ΔI and A are expressed, and upon I ₁. The equation is readily deduced on dimensional grounds, without reference to specific theories of the nature of ΔI or of retinal area in terms of its excitable units. Z is independent of the units of I and A. Experimentally it is found to be the same for monocular and binocular excitations, as is to be expected. Also as is expected it is not independent of λ, and it is markedly influenced by the scheme according to which A is varied; it depends directly upon the rate at which potentially excitable elements are added when A is made to increase. For simultaneous excitation of the two eyes (when of very nearly equivalent excitability), ΔĪ_B is less than for stimulation of either eye alone, at all levels of I ₁, A, λ. The mean ratio (ΔĪ_L + ΔĪ_R)/2 to ΔI^B was 1.38. For white light, doubling A on one retina reduces ΔI_m in the ratio 1.21, or a little less than for binocular presentation under the same conditions. These facts are consistent with the view that the properties of ΔI are quantitatively determined by events central to the retina. The measure σ_1ΔI of organic variation in discrimination of intensities and ΔI_m are found to be in simple proportion, independent of I ₁, A, λ (and exposure time). Variability (σ_1ΔI) is not a function of the mode of presentation, save that it may be slightly higher when both retinas are excited, and its magnitude (for a given level of ΔI_m) is independent of the law according to which the adjustable intensity I ₂ is instrumentally controlled.     

THEORY AND MEASUREMENT OF VISUAL MECHANISMS : X. MODIFICATIONS OF THE FLICKER RESPONSE CONTOUR,AND THE SIGNIFICANCE OF THE AVIAN PECTEN

1. When there is projected on the retina (man, monocularly) the shadow of a grid which forms a visual field in several distinct pieces (not including the fovea in the present tests), the ordinary properties of the flicker recognition contour (F vs. log I) as a function of the light-time cycle fraction (t_L) can be markedly disturbed. In the present experiments flicker was produced by the rotation of a cylinder with opaque vertical stripes. In the absence of such a grid shadow the "cone" segments of the contours form a set in which F_max. and the abscissa of inflection are opposite but rectilinear functions of t_L, while the third parameter of the probability integral (σ''_{log I}) remains constant. This is the case also with diverse other animals tested. In the data with the grid, however, analysis shows that even for low values of t_L (up to 0.50) there occurs an enhancement of the production of elements of neural effect, so that F_max. rises rather than falls as ordinarily with increase of t_L, although σ''_{log I} stays constant and hence the total number of acting units is presumed not to change. This constitutes valid evidence for neural integration of effects due to the illumination of separated retinal patches. Beginning at t_L = 0.75, and at 0.90, the slope of the "cone" curve is sharply increased, and the maximum F is far above its position in the absence of the grid. The decrease of σ''_{log I} (the slope constant) signifies, in terms of other information, an increase in the number of acting cone units. The abscissa of inflection is also much lowered, relatively, whereas without the grid it increases as t_L is made larger. These effects correspond subjectively to the fact that at the end-point flicker is most pronounced, on the "cone" curve, along the edges of the grid shadow where contrast is particularly evident with the longer light-times. The "rod" portion of the F - log I contour is not specifically affected by the presence of the grid shadow. Its form is obtainable at t_L = 0.90 free from the influence of summating "cone" contributions, because then almost no overlapping occurs. Analysis shows that when overlapping does occur a certain number of rod units are inhibited by concurrent cone excitation, and that the mean contribution of elements of neural action from each of the non-inhibited units is also reduced to an extent depending on the degree of overlap. The isolated "rod" curve at t_L = 0.90 is quite accurately in the form of a probability integral. The data thus give a new experimental proof of the occurrence of two distinct but interlocking populations of visual effects, and experimentally justify the analytical procedures which have been used to separate them. 2. The changing form of the F - log I contour as a function of t_L, produced in man when the illuminated field is divided into parts by a shadow pattern, is normally found with the bird Taeniopygia castenotis (Gould), the zebra finch. The retina has elements of one general structural type (cones), and the F - log I contour is a simplex symmetrical probability integral. The eye of this bird has a large, complex, and darkly pigmented pecten, which casts a foliated shadow on the retina. The change in form of the F - log I curve occurs with t_L above 0,50, and at t_L = 0.90 is quite extreme. It is more pronounced than the one that is secured in the human data with the particular grid we have used, but there is no doubt that it could be mimicked completely by the use of other grids. The increase of flicker acuity due to the pecten shadow is considerable, when the dark spaces are brief relative to the light. The evidence thus confirms the suggestion (Menner) drawn from comparative natural history that the visual significance of the avian pecten might be to increase the sensory effect of small moving images. It is theoretically important that (as in the human experiment) this may be brought about by an actual decrease of effective retinal area illuminated. It is also significant theoretically that despite the presence of shadows of pecten or of grid, and of the sensory influences thus introduced, the probability integral formulation remains effective.     

CAROTENOIDS AND THE VISUAL CYCLE

1. Carotenoids have been identified and their quantities measured in the eyes of several frog species. The combined pigment epithelium and choroid layer of an R. pipiens or esculenta eye contain about 1γ of xanthophyll and about 4γ of vitamin A. During light adaptation the xanthophyll content falls 10 to 20 per cent. 2. Light adapted retinas contain about 0.2–0.3 γ of vitamin A alone. 3. Dark adapted retinas contain only a trace of vitamin A. The destruction of their visual purple with chloroform liberates a hitherto undescribed carotenoid, retinene. The bleaching of visual purple to visual yellow by light also liberates retinene. Free retinene is removed from the isolated retina by two thermal processes: reversion to visual purple and decomposition to colorless products, including vitamin A. This is the source of the vitamin A of the light adapted retina. 4. Isolated retinas which have been bleached and allowed to fade completely contain several times as much vitamin A as retinas from light adapted animals. The visual purple system therefore expends vitamin A and is dependent upon the diet for its replacement. 5. Visual purple behaves as a conjugated protein in which retinene is the prosthetic group. 6. Vitamin A is the precursor of visual purple as well as the product of its decomposition. The visual processes therefore constitute a cycle.     

CRITICAL INTENSITY AND FLASH DURATION FOR RESPONSE TO FLICKER: WITH ANAX LARVAE

Determinations of the flicker response curve (F – log I_m) with larvae of Anax junius (dragonfly) for various ratios t_L/t_D of light time to dark time in a flash cycle provide relations between t_L/t_D and the parameters of the probability integral fundamentally describing the F – log I function, including the variability of I. These relations are quantitatively of the same form as those found for this function in the sunfish, and are therefore non-specific. Their meaning for the theory of reaction to visual flicker is discussed. The asymmetry of the Anax curve, resulting from mechanical conditions affecting the reception of light by the arthropod eye, is (as predicted) reduced by relative lengthening of the fractional light time in a cycle.     

THE FLICKER RESPONSE CONTOURS FOR GENETICALLY RELATED FISHES. II

The flicker response contour has been determined for several species and types of the teleosts Xiphophorus (X.) and Platypoecilius (P.) under the same conditions. The curve (F vs. log I_m) is the same for representatives of each generic type, but is different for the two genera. Its duplex nature is analyzable in each instance by application of the probability integral equation to the rod and cone constituent parts. The parameters of this function provide rational measures of invariant properties of the curves, which have specific values according to the genetic constitution of the animal. The F ₁ hybrids (H'''') of X. montezuma x P. variatus show dominance of the X. properties with respect to cone F_max. and σ'' _{log I}, but an intermediate value of the abscissa of inflection (τ''). The rod segment shows dominance of σ'' _{log I} from P., but an intermediate value of F_max. and of τ''. The composite flicker curve involves the operation of two distinct assemblages of excitable elements, differing quantitatively but not qualitatively in physicochemical organization, probably only secondarily related to the histological differentiation of rods and cones because almost certainly of central nervous locus, but following different rules in hereditary determination, and therefore necessarily different in physical organization. The interpretation of the diverse behavior of the three parameters of the probability summation is discussed, particularly in relation to the physical significance of these parameters as revealed by their quantitative relations to temperature, retinal area, and light time fraction in the flash cycle, and to their interrelations in producing the decline of rod effects at higher intensities. It is stressed that in general the properties of the parameters of a chosen interpretive analytical function must be shown experimentally to possess the physical properties implied by the equation selected before the equation can be regarded as describing those invariant properties of the organic system concerned upon which alone can deduction of the nature of the system proceed. The importance of genetic procedures in furthering demonstration that the biological performance considered in any particular case exhibits constitutionally invariant features provides a potentially powerful instrument in such rational analysis.     

The Role of Gene Duplication and Unconstrained Selective Pressures in the Melanopsin Gene Family Evolution and Vertebrate Circadian Rhythm Regulation

Melanopsin is a photosensitive cell protein involved in regulating circadian rhythms and other non-visual responses to light. The melanopsin gene family is represented by two paralogs, OPN4x and OPN4m, which originated through gene duplication early in the emergence of vertebrates. Here we studied the melanopsin gene family using an integrated gene/protein evolutionary approach, which revealed that the rhabdomeric urbilaterian ancestor had the same amino acid patterns (DRY motif and the Y and E conterions) as extant vertebrate species, suggesting that the mechanism for light detection and regulation is similar to rhabdomeric rhodopsins. Both OPN4m and OPN4x paralogs are found in vertebrate genomic paralogons, suggesting that they diverged following this duplication event about 600 million years ago, when the complex eye emerged in the vertebrate ancestor. Melanopsins generally evolved under negative selection (ω = 0.171) with some minor episodes of positive selection (proportion of sites = 25%) and functional divergence (θ_I = 0.349 and θ_II = 0.126). The OPN4m and OPN4x melanopsin paralogs show evidence of spectral divergence at sites likely involved in melanopsin light absorbance (200F, 273S and 276A). Also, following the teleost lineage-specific whole genome duplication (3R) that prompted the teleost fish radiation, type I divergence (θ_I = 0.181) and positive selection (affecting 11% of sites) contributed to amino acid variability that we related with the photo-activation stability of melanopsin. The melanopsin intracellular regions had unexpectedly high variability in their coupling specificity of G-proteins and we propose that Gq/11 and Gi/o are the two G-proteins most-likely to mediate the melanopsin phototransduction pathway. The selection signatures were mainly observed on retinal-related sites and the third and second intracellular loops, demonstrating the physiological plasticity of the melanopsin protein group. Our results provide new insights on the phototransduction process and additional tools for disentangling and understanding the links between melanopsin gene evolution and the specializations observed in vertebrates, especially in teleost fish.     

Primary Events, Energy Transfer, and Reactions in Photosynthetic Events: Lifetime of the Excited State In Vivo: II. Bacteriochlorophyll in Photosynthetic Bacteria at Room Temperature

Lifetime of the excited state (τ) of bacteriochlorophyll (BChl) in photosynthetic bacteria, measured with a mode-locked argon laser (oscillating at 488 nm; mode locked at 56 MHz) as light source, ranged from 0.3 to 2.5 nsec. These τ values are reported with a precision of ±0.1 nsec. The value of τ at high exciting light intensity (I) was two to three times that at low intensity. For young cultures of green bacterium Chloropseudomonas ethylicum, τ ranged from 0.5 (low I) to 1.0 nsec (high I); for those of the purple bacterium Rhodospirillum rubrum, from 0.4 (low I) to 1.0 nsec (high I); and for those of the BChl b-containing Rhodopseudomonas viridis, from 1.0 (low I) to 2.5 nsec (high I). These data provide information regarding the efficiencies of the photochemical process in these bacteria. Quantum yield (ø) of BChl fluorescence, calculated from ø = τ/τ₀ (where τ₀ is the intrinsic lifetime of fluorescence), ranges from 2-6% at low intensities to 6-14% at high intensities.     

Comparative Study of Action of Cell Wall Proteinases from Various Strains of Streptococcus cremoris on Bovine αs1-, β-, and κ-Casein

Partially purified cell wall proteinases of eight strains of Streptococcus cremoris were compared in their action on bovine α_s1-, β-, and κ-casein, as visualized by starch gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thin-layer chromatography. Characteristic degradation profiles could be distinguished, from which the occurrence of two proteinases, represented by strain HP and strain AM₁, was concluded. The action of the HP-type proteinase P₁ (also detectable in strains Wg₂, C₁₃, and TR) was established by electrophoretic methods to be directed preferentially towards β-casein. The AM₁-type proteinase P_III (also detectable in strain SK₁₁) was also able to degrade β-casein, but at the same time split α_s1- and κ-casein more extensively than did P_I. Strain FD₂₇ exhibited mainly P_I activity but also detectable P_III degradation characteristics. The cell wall proteinase preparation of strain E₈ showed low P_I as well as low P_III activity. All proteinase preparations produced from κ-casein positively charged degradation products with electrophoretic mobilities similar to those of degradation products released by the action of the milk-clotting enzyme chymosin. The differences between P_I and P_III in mode of action, as detected by gel electrophoresis and thin-layer chromatography, were reflected by the courses of the initial degradation of methyl-¹⁴C-labeled β-casein and by the effect of α_s1- plus κ-casein on these degradations. The results are discussed in the light of previous comparative studies of cell wall proteinases in strains of S. cremoris and with respect to the growth of this organism in milk.     

Synthesis,antioxidant and photoprotection activities of hybrid derivatives useful to prevent skin cancer

Chronic ultraviolet (UV) radiation exposure is a major cause of skin cancer. A novel series of hybrid derivatives (I–VIII) for use in sunscreen formulations were synthesized by molecular hybridization of t-resveratrol, avobenzone, and octyl methoxycinnamate, and were characterized. The antioxidant activity values for VIII were comparable than to those of t-resveratrol. Compounds I–III and VI demonstrated Sun Protector Factor superior to that of t-resveratrol. Compounds I and IV–VIII were identified as new, broad-spectrum UVA filters while II–III were UVB filters. In conclusion, novel hybrid derivatives with antioxidant effects have emerged as novel photoprotective agents for the prevention of skin cancer.     

A laboratory energy budget for the ormer Haliotis tuberculata L.

Each component of the energy budget (ingestion, egestion,somatic growth, reproductive investment, respiration, excretion, and mucus production) was measured for Haliotis tuberculata L. held at 15°C in a 12-h light: 12-h dark regime. Ulva lactuca L. was used as the food source throughout, and the budget was assessed over the whole size range of the animal. Ingestion rates ranged from 1.94 to 997.2 cal·animal⁻¹·day⁻¹ in 0.01 to 50 g dry wt (3.71 × 10⁻³ to 17.3 g dry flesh wt) animals, respectively. The major component of the energy budget was somatic growth (37.5% of I) in a 0.01-g dry wt animal while it was respiration (31.1% of I) in a 50-g dry wt animal. Mucus production formed a large part of the budget (from 23.3% of I in a 0.01-g dry wt animal to 29.l% of I in a 50-g dry wt animal). Scope for growth, I − (E + R + U + M), was calculated as ranging from 24.5% of ingestion in a 50-g dry wt animal to 36.8% in a 0.01-g dry wt animal.Each component was measured independently and allometric relationships with animal dry weight calculated. Exponents ranged from 0.60 (somatic growth) to 1.06 (reproductive investment).The calorific value of food was 3419 cal·g dry wt⁻¹ and for faeces was 2817 cal·g dry wt⁻¹. Absorption as a percentage of ingestion in terms of dry weight ranged from 78% for 95-mm length animals (50 g dry wt) to 81% in 6-mm animals (0.01-g dry wt⁻¹). Gross and net growth efficiencies (K₁ and K₂) were calculated on an energy basis and both were logarithmically related to animal dry weight.     

The initiator element of the Drosophila beta2 tubulin gene core promoter contributes to gene expression in vivo but is not required for male germ-cell specific expression

The tissue-specific expression of the Drosophila β2 tubulin gene (B2t) is accomplished by the action of a 14-bp activator element (β2UE1) in combination with certain regulatory elements of the TATA-less, Inr-containing B2t core promoter. We performed an in vivo analysis of the Inr element function in the B2t core promoter using a transgenic approach. Our experiments demonstrate that the Inr element acts as a functional cis-regulatory element in vivo and quantitatively regulates tissue-specific reporter expression in transgenic animals. However, our mutational analysis of the Inr element demonstrates no essential role of the Inr in mediating tissue specificity of the B2t promoter. In addition, a downstream element seems to affect promoter activity in combination with the Inr. In summary, our data show for the first time the functionality of the Inr element in an in vivo background situation in Drosophila.     

Cryptochrome, Phytochrome, and the Photoregulation of Anthocyanin Production under Blue Light

The principle of equivalent light action predicts that two light treatments (wavelengths ^λ₁ and λ₂) producing the same Pfr/P ratio (_λ1 = _λ2) and the same rate of phytochrome photoconversion (k_λ1 = k_λ2) are perceived by phytochrome as being the same and should produce the same effect. The results of experiments based on the principle of equivalent light action indicate that cryptochrome is involved in the photoregulation of anthocyanin production elicited by blue light in tomato seedlings. This was also the case for one strain of cabbage seedlings. For another strain of cabbage seedlings, the results suggest that cryptochrome is either not involved or that the state of phytochrome is the principal limiting factor.     

THE ORIENTATION OF ANIMALS BY OPPOSED BEAMS OF LIGHT

When orientation is attained under the influence of beams of parallel light opposed at 180° the deflection θ from a path at right angles to the beams is given by tan See PDF for Equation, where I ₁ and I ₂ are the photic intensities and H is the average angle between the photoreceptive surfaces. This expression is independent of the units in which I is measured, and holds whether the primary photosensory effect is proportional to I or to log I. When photokinetic side-to-side motions of the head occur, H decreases with increasing total acting light intensity, but increases if higher total light intensity restricts the amplitude of random movements; in each case, H is very nearly proportional to log I ₁ I ₂. For beams of light at 90°, See PDF for Equation. The application of these equations to some particular instances is discussed, and it is shown why certain simpler empirical formulæ previously found by others yield fair concordance with the experimental data. The result is thus in complete accord with the tropism theory, since the equations are based simply on the assumption that when orientation is attained photic excitation is the same on the two sides.     

Evolving visual pigments: Hints from the opsin-based proteins in a phylogenetically old “eyeless” invertebrate

Visual pigments are photosensitive receptor proteins that trigger the transduction process producing the visual excitation once they have absorbed photons. In spite of the molecular and morpho-functional complexity that has characterized the development of animal eyes and eyeless photoreceptive systems, opsin-based protein family appears ubiquous along metazoan visual systems. Moreover, in addition to classic rhodopsin photoreceptors, all Metazoa have supplementary non-visual photosensitive structures, mainly located in the central nervous system, that sense light without forming an image and that rather regulate the organism's temporal physiology. The investigation of novel non-visual photopigments exerting extraretinal photoreception is a challenging field in vision research. Here we propose the cnidarian Hydra as a useful tool of investigation for molecular and functional differences between these pigment families. Hydra is the first metazoan owning a nervous system and it is an eyeless invertebrate showing only an extraocular photoreception, as it has no recognized visual or photosensitive structures. In this paper we provide an overview of the molecular and functional features of the opsin-based protein subfamilies and preliminary evidences in a phylogenetically old species of both image-forming and non-visual opsins. Then we give new insights on the molecular biology of Hydra photoreception and on the evolutionary pathways of visual pigments.     

THE FLICKER RESPONSE CONTOUR FOR THE ISOPOD ASELLUS

The flicker response contour for the isopod Asellus is a simple probability integral (F - log I) over the whole determinable range (F = 1 to 51). This contrasts with the "distorted" asymmetrical curves obtained with Apis, Anax, and other arthropods with large convex eyes. The explanation of the distortion as due to mechanical conditions affecting photoreception is therefore confirmed, as the structure of the Asellus eye does not make such a factor likely to be expected for this case. The Asellus curve agrees with the only other available complete and uncomplicated flicker response contour (from Pseudemys, turtle with rod-free retina), in showing the superiority of the probability integral formulation as compared with certain others which have been suggested. It is noted as a curious and probably important fact that the relative dispersion of the intensity thresholds (σ''_{log I}) for the elements implicated in determining the flicker contour appears to be identical in bee, dragon fly nymph, and isopod. Other relevant information derived from similar experiments with vertebrates shows that this quantity is specifically determined by the organization of the animal. The nature of the common feature of neural organization in three such diverse arthropods, as contrasted with the diversity seen within one class of vertebrates (e.g., teleosts), remains to be discovered.