Characterization of bovine rod outer segment G-protein

Journal ArticleA simple modified procedure is described for isolating and purifying peripherally bound membrane proteins from bovine rod outer segment disks. The methods yield milligram quantities of G-protein and cGMP phosphodiesterase which are suitable for reconstitution with membranes containing visual pigments. The properties of cGMP phosphodiesterase have been previously characterized (Baehr, W., Devlin, M. J. & Applebury, M. L. (1979) J. Biol. Chem 254, 11669-11677). Here we report the characterization and properties of the rod outer segment G-protein, a complex of three subunits a, B, an

Surface modification of Ti-6Al-4V alloy for biomineralization and specific biological response: Part II, Alkaline phosphatase grafting

Titanium and its alloys are the most widespread materials for the realization of orthopaedic and dental implants due to their good mechanical properties and biocompatibility. Surface functionalization of biomaterials aimed to improve and quicken implant integration and tissue regeneration is an active research field. The opportunity to confer biological activity (ability to directly stimulate cells with proper biological signals) to the Ti6Al4 V alloy, previously modified to be bioactive from the inorganic point of view (apatite precipitation), was explored in this research work. The alkaline phosphatase (ALP) enzyme was grafted to metal surface via tresyl chloride activation, maintaining its activity. A synergistic effect between biological functionalization and inorganic bioactivity was observed

Analysis of Thyroid Response Element Activity during Retinal Development

Thyroid hormone (TH) signaling components are expressed during retinal development in dynamic spatial and temporal patterns. To probe the competence of retinal cells to mount a transcriptional response to TH, reporters that included thyroid response elements (TREs) were introduced into developing retinal tissue. The TREs were placed upstream of a minimal TATA-box and two reporter genes, green fluorescent protein (GFP) and human placental alkaline phosphatase (PLAP). Six of the seven tested TREs were first tested in vitro where they were shown to drive TH-dependent expression. However, when introduced into the developing retina, the TREs reported in different cell types in both a TH-dependent and TH-independent manner, as well as revealed specific spatial patterns in their expression. The role of the known thyroid receptors (TR), TRα and TRβ, was probed using shRNAs, which were co-electroporated into the retina with the TREs. Some TREs were positively activated by TR+TH in the developing outer nuclear layer (ONL), where photoreceptors reside, as well as in the outer neuroblastic layer (ONBL) where cycling progenitor cells are located. Other TREs were actively repressed by TR+TH in cells of the ONBL. These data demonstrate that non-TRs can activate some TREs in a spatially regulated manner, whereas other TREs respond only to the known TRs, which also read out activity in a spatially regulated manner. The transcriptional response to even simple TREs provides a starting point for understanding the regulation of genes by TH, and highlights the complexity of transcriptional regulation within developing tissue

Bat Eyes Have Ultraviolet-Sensitive Cone Photoreceptors

Mammalian retinae have rod photoreceptors for night vision and cone photoreceptors for daylight and colour vision. For colour discrimination, most mammals possess two cone populations with two visual pigments (opsins) that have absorption maxima at short wavelengths (blue or ultraviolet light) and long wavelengths (green or red light). Microchiropteran bats, which use echolocation to navigate and forage in complete darkness, have long been considered to have pure rod retinae. Here we use opsin immunohistochemistry to show that two phyllostomid microbats, Glossophaga soricina and Carollia perspicillata, possess a significant population of cones and express two cone opsins, a shortwave-sensitive (S) opsin and a longwave-sensitive (L) opsin. A substantial population of cones expresses S opsin exclusively, whereas the other cones mostly coexpress L and S opsin. S opsin gene analysis suggests ultraviolet (UV, wavelengths <400 nm) sensitivity, and corneal electroretinogram recordings reveal an elevated sensitivity to UV light which is mediated by an S cone visual pigment. Therefore bats have retained the ancestral UV tuning of the S cone pigment. We conclude that bats have the prerequisite for daylight vision, dichromatic colour vision, and UV vision. For bats, the UV-sensitive cones may be advantageous for visual orientation at twilight, predator avoidance, and detection of UV-reflecting flowers for those that feed on nectar

Diversity of Color Vision: Not All Australian Marsupials Are Trichromatic

Color vision in marsupials has recently emerged as a particularly interesting case among mammals. It appears that there are both dichromats and trichromats among closely related species. In contrast to primates, marsupials seem to have evolved a different type of trichromacy that is not linked to the X-chromosome. Based on microspectrophotometry and retinal whole-mount immunohistochemistry, four trichromatic marsupial species have been described: quokka, quenda, honey possum, and fat-tailed dunnart. It has, however, been impossible to identify the photopigment of the third cone type, and genetically, all evidence so far suggests that all marsupials are dichromatic. The tammar wallaby is the only Australian marsupial to date for which there is no evidence of a third cone type. To clarify whether the wallaby is indeed a dichromat or trichromatic like other Australian marsupials, we analyzed the number of cone types in the “dichromatic” wallaby and the “trichromatic” dunnart. Employing identical immunohistochemical protocols, we confirmed that the wallaby has only two cone types, whereas 20–25% of cones remained unlabeled by S- and LM-opsin antibodies in the dunnart retina. In addition, we found no evidence to support the hypothesis that the rod photopigment (rod opsin) is expressed in cones which would have explained the absence of a third cone opsin gene. Our study is the first comprehensive and quantitative account of color vision in Australian marsupials where we now know that an unexpected diversity of different color vision systems appears to have evolved

Light Perception in Two Strictly Subterranean Rodents: Life in the Dark or Blue?

BACKGROUND: The African mole-rats (Bathyergidae, Rodentia) are strictly subterranean, congenitally microphthalmic rodents that are hardly ever exposed to environmental light. Because of the lack of an overt behavioural reaction to light, they have long been considered to be blind. However, recent anatomical studies have suggested retention of basic visual capabilities. In this study, we employed behavioural tests to find out if two mole-rat species are able to discriminate between light and dark, if they are able to discriminate colours and, finally, if the presence of light in burrows provokes plugging behaviour, which is assumed to have a primarily anti-predatory function. METHODOLOGY/PRINCIPAL FINDING: We used a binary choice test to show that the silvery mole-rat Heliophobius argenteocinereus and the giant mole-rat Fukomys mechowii exhibit a clear photoavoidance response to full-spectrum ("white"), blue and green-yellow light, but no significant reaction to ultraviolet or red light during nest building. The mole-rats thus retain dark/light discrimination capabilities and a capacity to perceive short to medium-wavelength light in the photopic range of intensities. These findings further suggest that the mole-rat S opsin has its absorption maximum in the violet/blue part of the spectrum. The assay did not yield conclusive evidence regarding colour discrimination. To test the putative role of vision in bathyergid anti-predatory behaviour, we examined the reaction of mole-rats to the incidence of light in an artificial burrow system. The presence of light in the burrow effectively induced plugging of the illuminated tunnel. CONCLUSION/SIGNIFICANCE: Our findings suggest that the photopic vision is conserved and that low acuity residual vision plays an important role in predator avoidance and tunnel maintenance in the African mole-rats

Genetic Variations Strongly Influence Phenotypic Outcome in the Mouse Retina

Variation in genetic background can significantly influence the phenotypic outcome of both disease and non-disease associated traits. Additionally, differences in temporal and strain specific gene expression can also contribute to phenotypes in the mammalian retina. This is the first report of microarray based cross-strain analysis of gene expression in the retina investigating genetic background effects. Microarray analyses were performed on retinas from the following mouse strains: C57BL6/J, AKR/J, CAST/EiJ, and NOD.NON-H2-nb1 at embryonic day 18.5 (E18.5) and postnatal day 30.5 (P30.5). Over 3000 differentially expressed genes were identified between strains and developmental stages. Differential gene expression was confirmed by qRT-PCR, Western blot, and immunohistochemistry. Three major gene networks were identified that function to regulate retinal or photoreceptor development, visual perception, cellular transport, and signal transduction. Many of the genes in these networks are implicated in retinal diseases such as bradyopsia, night-blindness, and cone-rod dystrophy. Our analysis revealed strain specific variations in cone photoreceptor cell patterning and retinal function. This study highlights the substantial impact of genetic background on both development and function of the retina and the level of gene expression differences tolerated for normal retinal function. These strain specific genetic variations may also be present in other tissues. In addition, this study will provide valuable insight for the development of more accurate models for human retinal diseases