Early Cone Research Articles

STRA6 is critical for cellular vitamin A uptake and homeostasis.

Vitamin A must be adequately distributed within the body to maintain the functions of retinoids in the periphery and chromophore production in the eyes. Blood transport of the lipophilic vitamin is mediated by the retinol-binding protein, RBP4. Biochemical evidence suggests that cellular uptake of vitamin A from RBP4 is facilitated by a membrane receptor. This receptor, identified as the Stimulated by retinoic acid gene 6 (Stra6) gene product, is highly expressed in epithelia that constitute blood-tissue barriers. Here we established a Stra6 knockout mouse model to analyze the metabolic basis of vitamin A homeostasis in peripheral tissues. These mice were viable when bred on diets replete in vitamin A, but evidenced markedly reduced levels of ocular retinoids. Ophthalmic imaging and histology revealed malformations in the choroid and retinal pigmented epithelium, early cone photoreceptor cell death, and reduced lengths of rod outer segments. Similar to the blood-retina barrier in the RPE, vitamin A transport through the blood-cerebrospinal fluid barrier in the brain's choroid plexus was impaired. Notably, treatment with pharmacological doses of vitamin A restored vitamin A transport across these barriers and rescued the vision of Stra6(-/-) mice. Furthermore, under conditions mimicking vitamin A excess and deficiency, our analyses revealed that STRA6-mediated vitamin A uptake is a regulated process mandatory for ocular vitamin A uptake when RBP4 constitutes the only transport mode in vitamin A deficiency. These findings identifying STRA6 as a bona fide vitamin A transporter have important implications for disease states associated with impaired blood vitamin A homeostasis.

Crop damage caused by powdery mildew on hop and its relationship to late season management

Powdery mildew of hop (Podosphaera macularis) may cause economic loss due to reductions in cone yield and quality. Quantitative estimates of crop damage from powdery mildew remain poorly characterized, especially the effect of late season disease management on crop yield and quality. Field studies in Washington State evaluated cone yield, bittering acid content and quality factors when fungicide applications were ceased at different stages of cone development. The incidence of cones with powdery mildew was linearly correlated with yield of cones, bittering acids and accelerated cone maturation. In cultivar Galena, the cumulative effect of every 1% increase in cones powdery mildew incidence was to reduce alpha‐acid yield by 0·33%, which was due to direct effects on cone yield but also indirect effects mediated by dry matter. In the more susceptible cultivar Zeus, alpha‐acid yield was increased 20% by controlling powdery mildew through the transition of bloom to early cone development compared to ceasing fungicide applications at bloom: additional applications provided only modest improvements in alpha‐acid yield. In both cultivars, the impact of powdery mildew on aroma characteristics and bittering acid content were less substantial than cone yield. The damage caused by powdery mildew to cone colour and alpha‐acid yield, as well as the effectiveness of fungicide applications made to manage the disease, appears inseparably linked to dry matter content of cones at harvest. Realising achievable yield potential in these cultivars requires control of the disease through early stages of cone development and harvest before maturity exceeds c. 25% dry matter.

Fitting tips and tricks of challenging cases of keratoconus

AbstractPurpose to discuss the fitting tips and tricks of challenging cases of keratoconus with a special design of RGP lens namely Rose K2 lensMethods The study was carried on 52 keratoconic eyes fitted with Rose K2 lens. They were topographically classified into low cones, small steep cones, early cones, advanced cones and toric cones. The appropriate lens parameters were selected accordingly. Successful fitting was evaluated by lens position, movement, fluorescein pattern, visual acuity, residual astigmatism, patient comfort, maximum wearing time per day and corneal integrity.Results The keratoconic eyes were successfully fitted as follow: lenses with bigger diameter and considerably flatter base curve than normal were used to fit the low cones, lenses with flatter base curve, minimum diameter and maximum edge lift were used in small steep cones. Keeping the lens as small as possible and considerably steeper base curve than normal were used in advanced cones. Early cones were fitted with larger diameter lenses with decreased edge lift. Toric or astigmatic corneas were fitted acceptably either by reducing the overall diameter of the lens or using toric RGP lens. With the previous lens parameters, most of patients (96%) tolerated their lenses with maximum wearing time per day 11± 2.5 hours. No corneal staining or abrasion was encountered in studied eyes. Lenses were centrally positioned with adequate movement and acceptable fluorescein pattern. Best corrected visual acuity was significantly increased compared to the baseline visual acuity with significantly decreased astigmatism.Conclusion Patients with keratoconic corneas are sometimes challenging to fit and time consuming, but if well fitted with appropriate lens parameters, they appreciate the benefits provided by their contact lenses i

Otx2 and Onecut1 Promote the Fates of Cone Photoreceptors and Horizontal Cells and Repress Rod Photoreceptors

Cone photoreceptors carry out phototransduction in daylight conditions and provide the critical first step in color vision. Despite their importance, little is known about the developmental mechanisms involved in their generation, particularly how they are determined relative to rod photoreceptors, the cells that initiate vision in dim light. Here, we report the identification of a cis-regulatory module (CRM) for the thyroid hormone receptor beta (Thrb) gene, an early cone marker. We found that ThrbCRM1 is active in progenitor cells biased to the production of cones and an interneuronal cell type, the horizontal cell (HC). Molecular analysis of ThrbCRM1 revealed that it is combinatorially regulated by the Otx2 and Onecut1 transcription factors. Onecut1 is sufficient to induce cells with the earliest markers of cones and HCs. Conversely, interference with Onecut1 transcriptional activity leads to precocious rod development, suggesting that Onecut1 is critically important in defining cone versus rod fates.

Molecular control of normal and acrocona mutant seed cone development in Norway spruce (Picea abies) and the evolution of conifer ovule‐bearing organs

Reproductive organs in seed plants are morphologically divergent and their evolutionary history is often unclear. The mechanisms controlling their development have been extensively studied in angiosperms but are poorly understood in conifers and other gymnosperms. Here, we address the molecular control of seed cone development in Norway spruce, Picea abies. We present expression analyses of five novel MADS-box genes in comparison with previously identified MADS and LEAFY genes at distinct developmental stages. In addition, we have characterized the homeotic transformation from vegetative shoot to female cone and associated changes in regulatory gene expression patterns occurring in the acrocona mutant. The analyses identified genes active at the onset of ovuliferous and ovule development and identified expression patterns marking distinct domains of the ovuliferous scale. The reproductive transformation in acrocona involves the activation of all tested genes normally active in early cone development, except for an AGAMOUS-LIKE6/SEPALLATA (AGL6/SEP) homologue. This absence may be functionally associated with the nondeterminate development of the acrocona ovule-bearing scales. Our morphological and gene expression analyses give support to the hypothesis that the modern cone is a complex structure, and the ovuliferous scale the result of reductions and compactions of an ovule-bearing axillary short shoot in cones of Paleozoic conifers.

Early cone setting in Picea abies acrocona is associated with increased transcriptional activity of a MADS box transcription factor.

Conifers normally go through a long juvenile period, for Norway spruce (Picea abies) around 20 to 25 years, before developing male and female cones. We have grown plants from inbred crosses of a naturally occurring spruce mutant (acrocona). One-fourth of the segregating acrocona plants initiate cones already in their second growth cycle, suggesting control by a single locus. The early cone-setting properties of the acrocona mutant were utilized to identify candidate genes involved in vegetative-to-reproductive phase change in Norway spruce. Poly(A(+)) RNA samples from apical and basal shoots of cone-setting and non-cone-setting plants were subjected to high-throughput sequencing (RNA-seq). We assembled and investigated 33,383 expressed putative protein-coding acrocona transcripts. Eight transcripts were differentially expressed between selected sample pairs. One of these (Acr42124_1) was significantly up-regulated in apical shoot samples from cone-setting acrocona plants, and the encoded protein belongs to the MADS box gene family of transcription factors. Using quantitative real-time polymerase chain reaction with independently derived plant material, we confirmed that the MADS box gene is up-regulated in both needles and buds of cone-inducing shoots when reproductive identity is determined. Our results constitute important steps for the development of a rapid cycling model system that can be used to study gene function in conifers. In addition, our data suggest the involvement of a MADS box transcription factor in the vegetative-to-reproductive phase change in Norway spruce.

Visual photoreceptor subtypes in the chicken retina: melatonin-synthesizing activity and in vitro differentiation

The chicken retina contains five visual photoreceptor subtypes, based on the specific opsin gene they express. In addition to the central role they play in vision, some or all of these photoreceptors translate photoperiodic information into a day-night rhythm of melatonin production. This indolic hormone plays an important role in the photoperiodic regulation of retinal physiology. Previous studies have stopped short of establishing whether melatonin synthesis takes place in all the photoreceptor spectral subtypes. Another issue that has been left unsettled by previous studies is when during development are retinal precursor cells committed to a specific photoreceptor subtype and to a melatoninergic phenotype? To address the first question, in situ hybridization of the five opsins was combined with immunofluorescent detection of the melatonin-synthesizing enzyme hydroxyindole O-methyltransferase (HIOMT, EC.2.1.1.4). Confocal microscopy clearly indicated that all photoreceptor spectral subtypes are involved in melatonin synthesis. To tackle the second question, retinal precursor cells were dissociated between embryonic day 6 (E6) and E13 and cultured in serum-free medium for 4 days to examine their ability to autonomously activate the expression of opsins and HIOMT. Real-time PCR on cultured precursors indicated that red-, green- and violet-sensitive cones are committed at E6, rods at E10 and blue-sensitive cones at E12. HIOMT gene expression was programmed at E6, probably reflecting the differentiation of early cones. The present study provides a better characterization of photoreceptor subtypes in the chicken retina and describes a combination of serum-free culture and real-time PCR that should facilitate further developmental studies.

Gene therapy rescues cone structure and function in the 3-month-old rd12 mouse: a model for midcourse RPE65 leber congenital amaurosis.

RPE65 function is necessary in the retinal pigment epithelium (RPE) to generate chromophore for all opsins. Its absence results in vision loss and rapid cone degeneration. Recent Leber congenital amaurosis type 2 (LCA with RPE65 mutations) phase I clinical trials demonstrated restoration of vision on RPE65 gene transfer into RPE cells overlying cones. In the rd12 mouse, a naturally occurring model of RPE65-LCA early cone degeneration was observed; however, some peripheral M-cones remained. A prior study showed that AAV-mediated RPE65 expression can prevent early cone degeneration. The present study was conducted to test whether the remaining cones in older rd12 mice can be rescued. Subretinal treatment with the scAAV5-smCBA-hRPE65 vector was initiated at postnatal day (P)14 and P90. After 2 months, electroretinograms were recorded, and cone morphology was analyzed by using cone-specific peanut agglutinin and cone opsin-specific antibodies. Cone degeneration started centrally and spread ventrally, with cells losing cone-opsin staining before that for the PNA-lectin-positive cone sheath. Gene therapy starting at P14 resulted in almost wild-type M- and S-cone function and morphology. Delaying gene-replacement rescued the remaining M-cones, and most important, more M-cone opsin-positive cells were identified than were present at the onset of gene therapy, suggesting that opsin expression could be reinitiated in cells with cone sheaths. The results support and extend those of the previous study that gene therapy can stop early cone degeneration, and, more important, they provide proof that delayed treatment can restore the function and morphology of the remaining cones. These results have important implications for the ongoing LCA2 clinical trials.

Effect of lens diameter on lens performance and initial comfort of two types of GP lenses for keratoconus: a pilot study

Purpose The purpose of this pilot study was to determine the effect of varying lens diameter of two types of keratoconic lenses on lens performance and initial comfort with participants with either centered or oval cones. Methods Sixteen eyes of ten keratoconic participants were fitted with lenses of five diameters i.e. 8.7, 9, 9.6, 10.1 and 10.4 diameters and two commercially available lens types; “KCGP-1” and “KCGP-2”. Lensmovement, centration and initial comfort were assessed. Results Ten subjects (2 female and 8 male, sixteen eyes) were enrolled to participate in the pilot study, themean age was 40.4 ± 14.33 years. Six eyes were in the early centred cone group, five in the early oval cone group and five in the late oval cone group. The lenses with the 9.6 lens diameter (TD) decentered the least for all lenses (p = 0.001). When compared to cone type, the 8.7/9 were more decentered for the late oval and late centred cones (p = 0.009). The movement of the smaller KCGP-1 was greater than the KCGP-2 for the centered early cones (p = 0.001) and the movement decreased for the larger KCGP-2 lenses for all cone types but not significantly (p > 0.05). The KCGP-1 lenses were more significantly comfortable than the KCGP-2 lenses for the centered cones (p = 0.003). Only for the early oval cones, was the larger KCGP-2 lenses more comfortable (p = 0.04). Conclusions Lens diameter affects comfort and centration especially for the small (8.7/9) and large (10.4/10.1) diameters in this pilot study. Lens movement was not correlated with comfort.

Thyroid hormone accelerates opsin expression during early photoreceptor differentiation and induces opsin switching in differentiated TRα‐expressing cones of the salmonid retina

Thyroid hormone and its receptors (TRs) regulate photoreceptor differentiation and visual pigment protein (opsin) expression in the retinas of several vertebrates, including rodents and fish. In some of these animals, opsin expression can arise through switches within differentiated cone photoreceptors. In salmonid fishes, single cones express ultraviolet (SWS1) opsin during embryonic development and switch to blue (SWS2) opsin as the fishes grow. It is unknown whether thyroid hormone regulates opsin expression during early cone differentiation and acts through TRs to induce opsin switches in differentiated cones of the salmonid retina. Using in situ hybridization, we characterized the spatiotemporal dynamics of opsin expression and switching in embryos treated with exogenous thyroid hormone or propylthiouracil (PTU), a pharmacological inhibitor of thyroid hormone synthesis. We combined immunohistochemistry with in situ hybridization to map TRα expression with respect to cones undergoing the opsin switch in older juvenile fish. Thyroid hormone accelerated opsin expression in differentiating cones and induced the opsin switch in differentiated single cones, whereas PTU repressed the opsin switch. TRα was not detected in differentiating photoreceptors as opsin expression initiated, but was later expressed in differentiated single cones before the onset of the opsin switch. TRα expression exhibited a dynamic dorsoventral distribution that paralleled the progression of the opsin switch. Together, our results show that thyroid hormone is required for opsin switching in the retina of salmonid fishes and suggest that TRα may be involved in regulating this phenomenon.

S-opsin protein is incompletely modified during N-glycan processing in Rpe65−/− mice

Retinal pigment epithelium-specific protein 65 kDa (RPE65) is a key enzyme for the visual cycle in the eye. Rpe65 −/− mice lack 11- cis-retinal, and show early cone degeneration and mislocalization of cone opsins. The present study investigated whether abnormal modification of cone opsins at the protein level is present in Rpe65 −/− mice. Retina–RPE–choroids of Rpe65 −/− mice at 3, 5 and 7 weeks old were used. Immunohistochemistry of opsins was performed using cryosections and retinal flatmounts. We evaluated levels of mRNA for cone and rod opsin genes by RT-PCR and levels of proteins by western blotting. To examine modification patterns of N-glycan in Rpe65 −/− mice, cone opsins were digested with peptide-N-glycosidase (PNGase) F. S-opsin protein was detected at ∼40-kDa as a major band in wild-type mice, whereas ∼42-kDa S-opsin protein was detected in Rpe65 −/− mice. After PNGase F treatment, mobility of S-opsin protein in wild-type and Rpe65 −/− mice on SDS-PAGE was similar. In addition, ∼25-kDa S-opsin polypeptide was notably detected in Rpe65 −/− mice. Conversely, M-opsin proteins were not observed by immunohistochemistry or western blotting in Rpe65 −/− mice, but expression of M-opsin mRNA in Rpe65 −/− mice did not differ significantly from that in wild-type mice at 3 and 5 weeks. Mobility of M-opsin protein in Rpe65 −/− mice was unchanged. Our data suggest that S-opsin protein is incompletely modified during N-glycan processing in Rpe65 −/− mice, whereas M-opsin protein is severely reduced by posttranslational degradation in the absence of incomplete N-glycan processing in Rpe65 −/− mice.

Facies architecture of a Triassic rift-related Silicic Volcano-Sedimentary succession in the Tethyan realm, Peonias subzone, Vardar (Axios) Zone, northern Greece; Regional implications

In northern Greece, along the western edge of the Paleozoic Vertiscos terrane (Serbomacedonian massif) and within the Peonias subzone – the eastern part of the Vardar (Axios) Zone – a Silicic Volcano-Sedimentary (SVS) succession of Permo(?)–Skythian to Mid Triassic age records the development of a faulted continental margin and the formation of rhyolitic volcanoes along a continental shelf fringed by neritic carbonate accumulations. It represents the early rifting extensional stages that eventually led to the opening of the main oceanic basin in the western part of the Vardar (Axios) Zone (the Almopias Oceanic Basin). Even though the SVS succession is deformed, altered, extensively silicified and metamorphosed in the low greenschist facies, primary textures, original contacts and facies relationships are recognized in some places allowing clues for the facies architecture and the depositional environment. Volcanic and sedimentary facies analysis has been carried out at Nea Santa and Kolchida rhyolitic volcanic centres. Pyroclastic facies, mostly composed of gas-supported lapilli tuffs and locally intercalated accretionary lapilli tuffs, built the early cones which were then overridden by rhyolitic aphyric and minor K-feldspar–phyric lava flows. The characteristics of facies, especially the presence of accretionary lapilli, imply subaerial to coastal emplacement at this early stage. The mature and final stages of volcanism are mostly represented by quartz–feldspar porphyry intrusions that probably occupied the vents. At Nea Santa area, the presence of resedimented hyaloclastite facies indicates subaqueous emplacement of rhyolitic lavas and/or lobes. Moreover, quartz–feldspar–phyric sills and a partly extrusive dome featuring peperites at their margins are inferred to have intruded unconsolidated, wet carbonate sediments of the overlying Triassic Neritic Carbonate Formation, in a shallow submarine environment. The dome had probably reached above wave-base as is indicated by the presence of reworked rhyolitic clasts in the younger mixed rhyolite–carbonate epiclastic sedimentary facies. This facies is interpreted as mass- and debris-flow of mixed provenance, deposited below wave-base. The facies architecture of the SVS succession records a change in volcanic activity from explosive to effusive and then to intrusive. The depositional environment changed from subaerial-coastal to shallow submarine as the silicic volcanism evolved and carbonate sedimentation was progressively taking over, probably compensating for the gradual subsidence of the corresponding basin. Silicic magmatism and carbonate sedimentation were contemporaneous and spatially related. The timing of the rifting, the continental crustal elements involved and the accompanying tectonic, magmatic and sedimentary processes are features of the spatially and temporally evolving western peri-Tethyan region.

Selective heterogeneous oriented attachment of manganese oxide nanorods in water: toward 3D nanoarchitectures

MnOOH and MnO2 polymorphs are synthesized using a reaction between Mn2+and MnO4− in water at low temperature (95 °C). Nanoparticles are characterized using powder XRD, TEM and electron diffraction. Depending on the acidity of the aging medium, γ-MnO2 compounds are shown to be obtained as nanorods (final pH = 2.0) or hollow nanocones (3.6 ≤ final pH ≤ 4.5). The external faces of the cones originate from heterogeneous oriented attachment of α-MnOOH nanorods on early cones. The selective attachment between (−110)γ–MnO2 and (101)α–MnOOH faces is discussed by taking into account the surface charge of the primary particles and the reactivity of the Mn–OHx surface groups. This study underlines the role of the electrostatic repulsions and the surface reactivity in the oriented attachment mechanism in water for oxides.

Start of reproduction and allozyme heterozygosity in Pinus sibirica under different techniques of artificial forest stand establishment

Siberian stone pine (Pinus sibirica Du Tour) is one of the main forest-forming tree species in boreal forests of Eurasia. Large edible seeds of this species have an important resource value because of their high nutritious properties. Development of approaches to establishment of early cone producing Siberian stone pine stands including utilization of corresponding genetic background is one of the priorities of forest resource management. The goal of our study was to evaluate the effect of stand density on the differentiation of trees by the age of first reproduction and the relationship of allozyme heterozygosity and morphological traits variability in Siberian stone pine. Morphological and allozyme variability in artificial Pinus sibirica stands with high and low density was investigated. In the high-density stand the distance between trees was 0.7 and 3 meters (4080 trees per ha) while in the lowdensity stand it was 8 and 8 meters (144 trees per ha). Age of formation of first male and female cones was evaluated by retrospective method based on analysis of tracks of cones on a shoot bark. Tree height, diameter and number of male, female and vegetative shoots in a crown of model trees were measured. Genotypes of the trees were determined by 29 isozyme loci coding for 16 enzymes (ADH, FDH, FEST, GDH, GOT, IDH, LAP, MDH, MNR, PEPCA, 6-PGD, PGI, PGM, SDH, SKDH, SOD). In the low-density stand, the portion of generative trees was higher and differentiation of trees by age of reproduction starting was lower in spite of the smaller age of trees as compared to the high-density stand. In both samples, the age of formation of first generative organs was related negatively with stem height, stem diameter and number of female shoots. In the high-density stand, positive relation of age of first reproduction with total number of shoots and number of male shoots was found. In both samples nonreproductive trees were less heterozygous at isozyme loci as compared to trees starting cone production. Allozyme heterozygosity correlated positively with the age of first cone production in the high-density stand only. We conclude that the degree of competition between trees due to different stand density determined age and timing of first reproduction and development of tree crowns. The relationship of allozyme heterozygosity and the age of first reproduction was expressed more in the high-density stand of Siberian stone pine.

Human cone photoreceptor dependence on RPE65 isomerase

The visual (retinoid) cycle, the enzymatic pathway that regenerates chromophore after light absorption, is located primarily in the retinal pigment epithelium (RPE) and is essential for rod photoreceptor survival. Whether this pathway also is essential for cone photoreceptor survival is unknown, and there are no data from man or monkey to address this question. The visual cycle is naturally disrupted in humans with Leber congenital amaurosis (LCA), which is caused by mutations in RPE65, the gene that encodes the retinoid isomerase. We investigated such patients over a wide age range (3-52 years) for effects on the cone-rich human fovea. In vivo microscopy of the fovea showed that, even at the youngest ages, patients with RPE65-LCA exhibited cone photoreceptor loss. This loss was incomplete, however, and residual cone photoreceptor structure and function persisted for decades. Basic questions about localization of RPE65 and isomerase activity in the primate eye were addressed by examining normal macaque. RPE65 was definitively localized by immunocytochemistry to the central RPE and, by immunoblotting, appeared to concentrate in the central retina. The central retinal RPE layer also showed a 4-fold higher retinoid isomerase activity than more peripheral RPE. Early cone photoreceptor losses in RPE65-LCA suggest that robust RPE65-based visual chromophore production is important for cones; the residual retained cone structure and function support the speculation that alternative pathways are critical for cone photoreceptor survival.

Pathological and Electrophysiological Features of a Canine Cone–Rod Dystrophy in the Miniature Longhaired Dachshund

To characterize the electrophysiological and histopathological features of a retinal degenerative disease in a colony of miniature longhaired dachshunds known to have a form of progressive retinal atrophy (PRA). Serial electroretinograms were recorded from affected homozygous (n = 36) and heterozygous (n = 15) dogs. Morphologic investigations including immunohistochemistry and lectin histochemistry were performed on selected homozygous animals (n = 15). Clinical findings included loss of tapetal hyperreflectivity. The mode of inheritance was autosomal recessive. An early dramatic reduction of cone-specific ERG amplitude with a more modest reduction in rod b-wave amplitude was demonstrated. Progressively, rod specific responses diminished until there were no recordable responses to the ERG stimuli at 40 weeks of age. Morphologic changes confirmed early cone inner and outer segment loss. Other abnormalities included opsin mislocalization and outer nuclear layer thinning due to the subsequent loss of rod photoreceptors. A novel canine cone-rod dystrophy has been identified.

Eruptive and geomorphic processes at the Lathrop Wells scoria cone volcano

The ∼ 80 ka Lathrop Wells volcano (southern Nevada, U.S.A.) preserves evidence for a range of explosive processes and emplacement mechanisms of pyroclastic deposits and lava fields in a small-volume basaltic center. Early cone building by Strombolian bursts was accompanied by development of a fan-like lava field reaching ∼ 800 m distance from the cone, built upon a gently sloping surface. Lava flows carried rafts of cone deposits, which provide indirect evidence for cone facies in lieu of direct exposures in the active quarry. Subsequent activity was of a violent Strombolian nature, with many episodes of sustained eruption columns up to a few km in height. These deposited layers of scoria lapilli and ash in different directions depending upon wind direction at the time of a given episode, reaching up to ∼ 20 km from the vent, and also produced the bulk of the scoria cone. Lava effusion migrated from south to north around the eastern base of the cone as accumulation of lavas successively reversed the topography at the base of the cone. Late lavas were emplaced during violent Strombolian activity and continued for some time after explosive eruptions had waned. Volumes of the eruptive products are: fallout — 0.07 km 3, scoria cone — 0.02 km 3, and lavas — 0.03 km 3. Shallow-derived xenolith concentrations suggest an upper bound on average conduit diameter of ∼ 21 m in the uppermost 335 m beneath the volcano. The volcano was constructed over a period of at least seven months with cone building occurring only during part of that time, based upon analogy with historical eruptions. Post-eruptive geomorphic evolution varied for the three main surface types that were produced by volcanic activity: (1) scoria cone, (2) low relief surfaces (including lavas) with abundant pyroclastic material, and (3) lavas with little pyroclastic material. The role of these different initial textures must be accounted for in estimating relative ages of volcanic surfaces, and failure to account for this resulted in previous erroneous interpretation that the volcano's activity was polycyclic (eruptions separated by 1000s–10,000s of years). Lathrop Wells volcano provides an example of the wide range of eruptive processes that can occur with little change in major element composition; the variation in explosive and effusive processes, including their simultaneous occurrence, must result entirely from fluid dynamic, crystallization, and degassing processes in the ascending multiphase magma. The volcano also provides key analog information regarding processes that are important for volcanic risk assessment at the proposed Yucca Mountain radioactive waste repository, ∼ 18 km north of the volcano.

Cell Cycle–Specific and Cell Type–Specific Expression of Rb in the Developing Human Retina

To define the pattern of Rb expression relative to cell cycle position and cell type in the developing human retina. Cryosections of fetal week 11-18 retinas were immunostained for Rb and cell cycle- or cell type-specific markers. Rb was prominent in retinal progenitor cells (RPCs) expressing the cyclin D1, cyclin A, and cytoplasmic cyclin B markers of G1, S, and early to mid G2 phases, but not in RPCs expressing the phosphohistone H3 marker of late G2 and M. Rb was not detected in the earliest postmitotic ganglion, amacrine, horizontal, and bipolar cell precursors migrating away from the ventricular layer, but was detected as such cells underwent further differentiation. Among photoreceptors, Rb was not detected in the earliest RXRgamma(+) cone precursors or in the earliest Nrl(+) rod precursors, but subsequently rose to high levels in cones and to low levels in rods. Rb was prominent at the time when Müller glia exit the cell cycle and was generally expressed in a pattern complementary to p27(Kip1). Rb exhibits cell cycle-specific expression in RPCs, with loss in late G2-M and restoration in G1. Rb is re-expressed after postmitotic ganglion, amacrine, horizontal, and bipolar cell precursors migrate away from the ventricular layer; after the appearance of early cone and rod markers; but coinciding with Müller glia cell cycle withdrawal. The results suggest that Rb does not mediate the initial proliferative arrest of retinal neurons, but may indirectly induce arrest in RPCs or maintain an arrest in postmitotic precursors.

Two Point Mutations of RPE65 from Patients with Retinal Dystrophies Decrease the Stability of RPE65 Protein and Abolish Its Isomerohydrolase Activity

RPE65 is the isomerohydrolase in the retinoid visual cycle essential for recycling of 11-cis retinal, the chromophore for visual pigments in both rod and cone photoreceptors. Mutations in the RPE65 gene are associated with inherited retinal dystrophies with unknown mechanisms. Here we show that two point mutations of RPE65, R91W and Y368H, identified in patients with retinal dystrophies both abolished the isomerohydrolase activity of RPE65 after a subretinal injection into the Rpe65-/- mice and in the in vitro isomerohydrolase activity assay, independent of their protein levels. Further, the R91W and Y368H mutants showed significantly decreased protein levels but unchanged mRNA levels when compared with the wild-type RPE65 (wtRPE65). Protein stability analysis showed that wtRPE65 is a fairly stable protein, with an apparent half-life longer than 10 h, when expressed in 293A cells. Under the same conditions, mutants R91W and Y368H both showed substantially decreased protein stabilities, with half-lives less than 2 and 6 h, respectively. Subcellular fractionation and Western blot analysis demonstrated that wtRPE65 predominantly exists in the membrane fraction, while both of the mutants are primarily distributed in the cytosolic fraction, suggesting that these mutations disrupt the membrane association of RPE65. However, palmitoylation assay showed that wtRPE65 and both of the mutants were palmitoylated. These results suggest that these mutations may result in critical structural alterations of RPE65 protein, disrupt its membrane association, and consequently impair its isomerohydrolase activity, leading to retinal degeneration.

RPE65Gene Delivery Restores Isomerohydrolase Activity and Prevents Early Cone Loss inRpe65−/−Mice

Recent in vitro evidence has shown that RPE65 is the isomerohydrolase that converts all-trans retinyl ester to 11-cis retinal, the chromophore for visual pigments in vertebrates. Homozygous RPE65 knockout (Rpe65-/-) mice lack 11-cis retinoids and have early cone degeneration. The purpose of this study is to determine whether RPE65 gene delivery restores the isomerohydrolase activity and normal profile of endogenous retinoids in Rpe65-/- mice. Adenovirus-expressing RPE65 (Ad-RPE65) was injected into the subretinal space of Rpe65-/- mice. The expression of RPE65 was determined by immunohistochemistry and Western blot analysis. The isomerohydrolase activity was measured in vitro in eyecup homogenates. Endogenous retinoid profile in the eyecups was analyzed by high-performance liquid chromatography (HPLC). Photoreceptor-specific gene expression was determined with real-time RT-PCR. Cone degeneration was determined by cone-specific staining and counting cones in flatmounted retina. High levels of RPE65 expression from the Ad-RPE65 injection generated robust isomerohydrolase activity in the eyecup of Rpe65-/- mice, at levels comparable to those in wild-type (wt) mice. Consequently, the RPE65 gene delivery resulted in substantial amounts of 11-cis retinal in Rpe65-/- mice. The RPE65 gene delivery prevented the downregulation of cone-specific genes, including both cone opsins and cone tranducin alpha subunit in Rpe65-/- mice. Moreover, the Ad-RPE65 injection also prevented massive cone degeneration at early ages of Rpe65-/- mice. RPE65 gene delivery generates isomerohydrolase activity and restores retinoid profile in Rpe65-/- mice. Regeneration of 11-cis retinal is essential for survival of cone photoreceptors.