Control of Retinal Sensitivity : I. Light and Dark Adaptation of Vertebrate Rods and Cones

Rods and cones in Necturus respond with graded hyperpolarization to test flashes spanning about 3.5 log units of intensity. Steady background levels hyperpolarize the rods, and the rod responses become progressively smaller as background level is increased. In cones, higher background levels reduce the effectiveness of test flashes, so higher ranges of test intensities are required to elicit the full range of graded responses. When backgrounds are terminated, cones return rapidly, but rods return slowly to the dark potential level. The effects of backgrounds on both rods and cones can be observed at intensities that cause negligible bleaching as determined by retinal densitometry. During dark adaptation, changes are observed in the rods and cones that are similar to those produced by backgrounds. Receptor sensitivities, derived from these results, show that rods saturate, cones obey Weber's law, and sensitization during dark adaptation follows a two-phase time-course.     

Age-Related Retinal Degeneration in Animal Models of Aging: Possible Involvement of Taurine Deficiency and Oxidative Stress

There is strong evidence that the retina degenerates with age. Electroretinogram deficits and photoreceptor cell death and structural abnormalities have been observed in both animal and human studies of aging. The mechanism behind this phenomenon is a very interesting area for scientific and medical study. Current data support the link between retinal degeneration and increased oxidative stress. Taurine is a free amino acid found in high millimolar concentrations in the retina, and age-related deficiency in retinal levels of taurine may contribute to the retinal degeneration associated with age. Taurine acts as an antioxidant and taurine replenishment is known to alleviate oxidative stress in the retina. Thus taurine supplementation may be useful in the treatment of age-related retinal dysfunction.     

Visible Light Induces Melanogenesis in Human Skin through a Photoadaptive Response

Visible light (400–700 nm) lies outside of the spectral range of what photobiologists define as deleterious radiation and as a result few studies have studied the effects of visible light range of wavelengths on skin. This oversight is important considering that during outdoors activities skin is exposed to the full solar spectrum, including visible light, and to multiple exposures at different times and doses. Although the contribution of the UV component of sunlight to skin damage has been established, few studies have examined the effects of non-UV solar radiation on skin physiology in terms of inflammation, and limited information is available regarding the role of visible light on pigmentation. The purpose of this study was to determine the effect of visible light on the pro-pigmentation pathways and melanin formation in skin. Exposure to visible light in ex-vivo and clinical studies demonstrated an induction of pigmentation in skin by visible light. Results showed that a single exposure to visible light induced very little pigmentation whereas multiple exposures with visible light resulted in darker and sustained pigmentation. These findings have potential implications on the management of photo-aggravated pigmentary disorders, the proper use of sunscreens, and the treatment of depigmented lesions.     

Dark Current and Photocurrent in Retinal Rods

The interstitial voltages, currents, and resistances of the receptor layer of the isolated rat retina have been investigated with arrays of micropipette electrodes inserted under direct visual observation by infrared microscopy. In darkness a steady current flows inward through the plasma membrane of the rod outer segments. It is balanced by equal outward current distributed along the remainder of each rod. Flashes of light produce a photocurrent which transiently reduces the dark current with a waveform resembling the PII and a-wave components of the electroretinogram. The photocurrent is produced by a local action of light within 12 μm of its point of absorption in the outer segments. The quantum current gain of the photocurrent is greater than 10⁶. The electrical space constant of rat rods is greater than 25 μm, so that the electrical effects of the photocurrent are large enough at the rod synapses to permit single absorbed photons to be detected by the visual system. The photocurrent is apparently the primary sensory consequence of light absorption by rhodopsin.     

Differential Expression of mRNA and Protein Encoding Retinal and Pineal S-Antigen During the Light/Dark Cycle

S-Antigen is a soluble cell protein unique to the retina and pineal gland. In the former, it is a well-characterized molecule that participates in light-induced signal transduction in photoreceptor cells. In the latter, the functional role is presently not known. The expression of S-antigen and its mRNA was examined in the rat retina and pineal gland throughout the diurnal cycle and with light interruption of the dark cycle. A cDNA for rat S-antigen was isolated from a pineal gland library to examine the mRNAs. A 1.7-kb mRNA for S-antigen was observed in both the pineal gland and the retina. Retinal S-antigen mRNA was expressed throughout the diurnal cycle and increased with light interruption of the dark cycle. In contrast, pineal gland S-antigen mRNA levels were detectable only during the dark and were absent preceding and during light. The phenotypic expression of immunoreactive S-antigen, identified with two S-antigen monoclonal antibodies (MAbs), MAb A9C6 and MAb C10C10, was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and isoelectric focusing (IEF) electrophoresis. Immunoblot analysis of gels after SDS-PAGE revealed a single 46-kDa protein in retina. In contrast, two bands of approximately 43 and 46 kDa were identified in the pineal gland. Immunoblots of the retinal extracts separated by IEF electrophoresis revealed five S-antigen isomers, which vary quantitatively throughout the diurnal cycle and when light interrupted the dark cycle. Immunoblots of the pineal gland samples separated by IEF electrophoresis indicated that the pineal gland possesses four pineal gland-specific forms of S-antigen in addition to the five forms present in the retina. The differences observed in the mRNA and protein analyses suggest tissue-specific structural components for S-antigen in the retina and pineal gland that are not regulated in the same manner.     

Postnatal Constant Light Compensates Cryptochrome1 and 2 Double Deficiency for Disruption of Circadian Behavioral Rhythms in Mice under Constant Dark

Clock genes Cryptochrome (Cry1) and Cry2 are essential for expression of circadian rhythms in mice under constant darkness (DD). However, circadian rhythms in clock gene Per1 expression or clock protein PER2 are detected in the cultured suprachiasmatic nucleus (SCN) of neonatal Cry1 and Cry2 double deficient (Cry1 ^-/-/Cry2 ^-/-) mice. A lack of circadian rhythms in adult Cry1 ^-/-/Cry2 ^-/- mice is most likely due to developmentally disorganized cellular coupling of oscillating neurons in the SCN. On the other hand, neonatal rats exposed to constant light (LL) developed a tenable circadian system under prolonged LL which was known to fragment circadian behavioral rhythms. In the present study, Cry1 ^-/-/Cry2 ^-/- mice were raised under LL from postnatal day 1 for 7 weeks and subsequently exposed to DD for 3 weeks. Spontaneous movement was monitored continuously after weaning and PER2::LUC was measured in the cultured SCN obtained from mice under prolonged DD. Surprisingly, Chi square periodogram analysis revealed significant circadian rhythms of spontaneous movement in the LL-raised Cry1 ^-/-/Cry2 ^-/- mice, but failed to detect the rhythms in Cry1 ^-/-/Cry2 ^-/- mice raised under light-dark cycles (LD). By contrast, prolonged LL in adulthood did not rescue the circadian behavioral rhythms in the LD raised Cry1 ^-/-/Cry2 ^-/- mice. Visual inspection disclosed two distinct activity components with different periods in behavioral rhythms of the LL-raised Cry1^-/-/Cry2^-/- mice under DD: one was shorter and the other was longer than 24 hours. The two components repeatedly merged and separated. The patterns resembled the split behavioral rhythms of wild type mice under prolonged LL. In addition, circadian rhythms in PER2::LUC were detected in some of the LL-raised Cry1^-/-/Cry2^-/- mice under DD. These results indicate that neonatal exposure to LL compensates the CRY double deficiency for the disruption of circadian behavioral rhythms under DD in adulthood.     

Dark Adaptation and Visual Pigment Regeneration in Human Cones

Foveal threshold elevation and red-green cone pigment regeneration have been studied in the dark after a wide range of bleaches in normal man with a view to probing the limits of the application of the Dowling-Rushton relation: i.e., the direct proportionality between log threshold elevation and fraction of unregenerated pigment. Cone pigment regeneration (and threshold recovery) is much faster after short bleaches than expected from the kinetics of a simple monomolecular reaction. Recovery is faster after a fixed (short) duration bleach the weaker it is. Except for the first 30 s after relatively weak bleaches and the entire recovery after a very brief (<0.001 s) saturating bright flash which bleaches a little more than 50%, the results are accurately fit by the Dowling-Rushton relation over the entire range tested with only one arbitrary constant (the proportionality factor). Theory predicts too low threshold in comparison with what is obtained, for both of these exceptions     

Phosphorylation and Dephosphorylation of Guard-Cell Proteins from Vicia faba L. in Response to Light and Dark

Phosphorylation and dephosphorylation of proteins were investigated in guard-cell protoplasts from Vicia faba L. When guard-cell protoplasts were incubated with 32Pi in the dark for 80 min, several proteins, with molecular masses of 42, 40, 34, 32, 26, and 19 kD, were phosphorylated. Illumination of the dark-adapted protoplasts with red light caused dephosphorylation of the 26-kD protein, but there was no detectable change in levels of phosphorylation in other proteins. In the dephosphorylation of the 26-kD protein, far-red light of 730 nm was most effective, but when the light was turned off, the protein was phosphorylated to the original level within 10 min. Subcellular fractionation of guard-cell protoplasts indicated that the 26-kD protein was located in the chloroplast. The migration pattern of the 26-kD protein was exactly the same as the light-harvesting Chl a/b protein complex of photosystem II (LHCPII) from Vicia mesophyll cells on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dephosphorylated 26-kD protein was phosphorylated by adding sodium hydrosulfite, a strong reducing agent, under the far-red illumination of guard-cell protoplasts. The magnitude of dephosphorylation by red light (660 nm) was increased by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an electron transfer inhibitor of photosystem II (PSII). Light-induced dephosphorylation was inhibited by 1 nM okadaic acid, an inhibitor of serine/threonine protein phosphatase. From these results, it is concluded that the 26-kD protein is LHCPII and that LHCPII is present mostly in the phosphorylated form in the dark and is dephosphorylated by type 2A protein phosphatase under the light absorbed by photosystem I in Vicia guard-cell protoplasts.     

Apo-Opsin and Its Dark Constitutive Activity across Retinal Cone Subtypes

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p53 Stabilization Induces Cell Growth Inhibition and Affects IGF2 Pathway in Response to Radiotherapy in Adrenocortical Cancer Cells

Adrenocortical carcinoma (ACC) is a very rare endocrine tumour, with variable prognosis, depending on tumour stage and time of diagnosis. However, it is generally fatal, with an overall survival of 5 years from detection. Radiotherapy usefulness for ACC treatment has been widely debated and seems to be dependent on molecular alterations, which in turn lead to increased radio-resistance. Many studies have shown that p53 loss is an important risk factor for malignant adrenocortical tumour onset and it has been reported that somatic mutations in TP53 gene occur in 27 to 70% of adult sporadic ACCs. In this study, we investigated the role of somatic mutations of the TP53 gene in response to ionizing radiation (IR). We studied the status of p53 in two adrenocortical cell lines, H295R and SW-13, harbouring non-functioning forms of this protein, owing to the lack of exons 8 and 9 and a point mutation in exon 6, respectively. Moreover, these cell lines show high levels of p-Akt and IGF2, especially H295R. We noticed that restoration of p53 activity led to inhibition of growth after transient transfection of cells with wild type p53. Evaluation of their response to IR in terms of cell proliferation and viability was determined by means of cell count and TUNEL assay.^wtp53 over-expression also increased cell death by apoptosis following radiation in both cell lines. Moreover, RT-PCR and Western blotting analysis of some p53 target genes, such as BCL2, IGF2 and Akt demonstrated that p53 activation following IR led to a decrease in IGF2 expression. This was associated with a reduction in the active form of Akt. Taken together, these results highlight the role of p53 in response to radiation of ACC cell lines, suggesting its importance as a predictive factor for radiotherapy in malignant adrenocortical tumours cases.     

Phototropism, Adaptation, and the Light-Growth Response of Phycomyces

Phototropic bending can be initiated without the transient changes in growth speed that characterize a light-growth response. The conditions required are a change from a symmetric to an asymmetric illumination pattern while the cell receives a constant radiant flux. Phototropism is thus basically a steady state process. It cannot be founded on differential light-growth responses as in Blaauw's theory. A possible model system for the unequal partition of growth during steady bending is discussed. The fact that light-growth responses show adaptation while phototropic bending does not follows from the different natures of the two responses.     

Zinc Deficiency Induces Oxidative Damage and Causes Spleen Fibrosis

Biological Trace Element Research - Zinc (Zn) is an essential trace element for animals. Zn controls the action of more than 300 enzymes and plays an important role in the regulation of gene...     

Spermidine Feeding Decreases Age-Related Locomotor Activity Loss and Induces Changes in Lipid Composition

Spermidine is a natural polyamine involved in many important cellular functions, whose supplementation in food or water increases life span and stress resistance in several model organisms. In this work, we expand spermidine’s range of age-related beneficial effects by demonstrating that it is also able to improve locomotor performance in aged flies. Spermidine’s mechanism of action on aging has been primarily related to general protein hypoacetylation that subsequently induces autophagy. Here, we suggest that the molecular targets of spermidine also include lipid metabolism: Spermidine-fed flies contain more triglycerides and show altered fatty acid and phospholipid profiles. We further determine that most of these metabolic changes are regulated through autophagy. Collectively, our data suggests an additional and novel lipid-mediated mechanism of action for spermidine-induced autophagy.     

Complement C3 and C5 Deficiency Affects Fracture Healing

There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3^-/- and C5^-/- as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3^-/-: -25%, p=0.02; C5^-/-: -20% p=0.052) and newly formed bone (C3^-/-: -38%, p=0.01; C5^-/-: -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3^-/- mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.     

LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

Actin exists as a monomer (G-actin) which can be polymerized to filaments) F-actin) that under the influence of actin-binding proteins and polycations bundle and contribute to the formation of the cytoskeleton. Bundled actin from lysed cells increases the viscosity of sputum in lungs of cystic fibrosis patients. The human host defense peptide LL-37 was previously shown to induce actin bundling and was thus hypothesized to contribute to the pathogenicity of this disease. In this work, interactions between actin and the cationic LL-37 were studied by optical, proteolytic and surface plasmon resonance methods and compared to those obtained with scrambled LL-37 and with the cationic protein lysozyme. We show that LL-37 binds strongly to CaATP-G-actin while scrambled LL-37 does not. While LL-37, at superstoichiometric LL-37/actin concentrations polymerizes MgATP-G-actin, at lower non-polymerizing concentrations LL-37 inhibits actin polymerization by MgCl₂ or NaCl. LL-37 bundles Mg-F-actin filaments both at low and physiological ionic strength when in equimolar or higher concentrations than those of actin. The LL-37 induced bundles are significantly less sensitive to increase in ionic strength than those induced by scrambled LL-37 and lysozyme. LL-37 in concentrations lower than those needed for actin polymerization or bundling, accelerates cleavage of both monomer and polymer actin by subtilisin. Our results indicate that the LL-37-actin interaction is partially electrostatic and partially hydrophobic and that a specific actin binding sequence in the peptide is responsible for the hydrophobic interaction. LL-37-induced bundles, which may contribute to the accumulation of sputum in cystic fibrosis, are dissociated very efficiently by DNase-1 and also by cofilin.