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J C Besharse

Publications and source records attributed to J C Besharse.

At least 73 records · Page 4Linked to original sources
PubMed1985

Light and temperature modulated staining of the rod outer segment distal tips with Lucifer yellow.

External application of the dye Lucifer yellow to isolated retinas of Xenopus laevis causes a specific staining of the distal tips of the rod outer segment (ROS). Staining occurs most frequently in the distal 4 micron of the ROS and does not diffuse throughout the ROS cytosol. In contrast, damaged ROS fill with the dye and exhibit a diffuse fluorescence. Retinas from constant light-treated animals show a greater frequency of labeling when isolated in the light than when isolated in the dark after 0.5 and 3 hr. Increased frequency of distal tip staining for dark-treated animals can be achieved if animals are returned to the light. Distal tip labeling also occurs in cyclic-light maintained animals but at a much lower frequency. The frequency of distal tip staining can also be altered by temperature and metabolic poisons. Isolated retinas exposed to dye solutions kept at either 3 degrees C or containing the metabolic poisons, iodoacetate or dinitrophenol, exhibited a reduced frequency of staining. This suggests that staining is an active process involving cellular metabolism. The distal location, dimensions, and light dependence of staining suggests that labeled regions are destined for detachment as part of disc shedding. The sensitivity of distal tip staining to metabolic poisons suggests that the photoreceptor plays a role in determining the membrane domains destined for shedding.

Animals↗
PubMed1985

Vesicular transport of newly synthesized opsin from the Golgi apparatus toward the rod outer segment. Ultrastructural immunocytochemical and autoradiographic evidence in Xenopus retinas.

Each day, rod photoreceptors of the vertebrate retina synthesize rhodopsin and insert it into new membranes of the rod outer segment (ROS). The authors determined which components of the rod cell transport opsin from the Golgi to the ROS by a combined EM autoradiographic and immunocytochemical study using radiolabeled amino acid precursors and antiopsin antibodies. Radiolabeled proteins in the ellipsoid region of Xenopus laevis retinal rods were localized by comparison of the distribution of silver grains with the predicted distribution generated by a hypothetical source: grain matrix. Sources of decay were not uniformly distributed. Small vesicles compressed between mitochondria and clustered beneath the connecting cilium that joins the inner to the outer segment contained more than 30% of the radiolabel and had a specific activity 17 times higher than the surrounding cytoplasm. Opsin was localized immunocytochemically on thin sections of retinas embedded in Lowicryl K4M (Polysciences; Warrington, PA) by reaction sequentially with biotinyl-rabbit antifrog opsin, biotinyl-sheep antirabbit F(ab')2, and avidin-ferritin. Golgi apparatus, intermitochondrial vesicles, and vesicles that clustered beneath the connecting cilium were prominently labeled. Subellipsoid smooth endoplasmic reticulum was labeled at background levels. These results demonstrate that intracellular vesicular membranes transport newly synthesized opsin from the Golgi to the base of the connecting cilium of X. laevis retinas. Antibody labeled the outer segment plasma membrane at a 10-fold greater density than the contiguous inner segment plasma membrane. The polarized distribution of opsin apparently involves not only vectorial transport of opsin in the inner segment but also restrictions to the randomization of opsin inserted into the inner and outer segment plasma membrane.

Animals↗
PubMed1985

Membrane assembly in retinal photoreceptors. II. Immunocytochemical analysis of freeze-fractured rod photoreceptor membranes using anti-opsin antibodies.

We have used a cytochemical technique for labeling freeze-fractured tissues (Pinto da Silva, P., C. Parkison, and N. Dwyer (1981) Proc. Natl. Acad. Sci. U.S.A. 78: 343-347) to examine the distribution of immunoreactive opsin in rod photoreceptor membranes. Aldehyde-fixed retinas of African clawed frogs (Xenopus laevis) embedded in a cross-linked protein matrix were frozen and fractured at -196 degrees C, then thawed and labeled with biotinylated sheep anti-cow opsin IgG followed by avidin-ferritin. In thin sections of plastic-embedded retinas, rod outer segment (ROS) disc membranes exposed by fracturing bound specific antibody intensely and relatively uniformly. However, they differed from membranes of the inner segment as well as those of erythrocytes in that protoplasmic face leaflets did not assume an interrupted bilayer appearance and disc exoplasmic face leaflets were apparently lost during thawing. The disposition of opsin immunoreactivity in the cell membrane was highly asymmetric. Although ROS plasma membranes from which discs are elaborated labeled heavily with anti-opsin after cleavage, fractures passing along inner segment plasma membranes bound very little antibody. In cross-fractures exposing inner segment cytoplasm, we found specific labeling of Golgi complex elements, as well as both perimitochondrial and periciliary vesicles. The latter are presumed to be the vehicle shuttling newly synthesized membrane to the ROS for disc assembly. These results suggest that opsin-containing membrane is sorted out within the cell, being transported from synthetic sites to the immediate periciliary zone where localized insertion into the cell membrane takes place. Furthermore, the close correspondence of the present immunocytochemical analysis with the distribution of opsin deduced from prior quantitative freeze-fracture analysis (Besharse, J. C., and K. H. Pfenninger (1980) J. Cell Biol. 87: 451-463) offers the possibility that fracture-label may be generally useful for study of patterned membrane topography in neuronal cells.

Animals↗
PubMed1985

Membrane assembly in retinal photoreceptors. III. Distinct membrane domains of the connecting cilium of developing rods.

To investigate the putative role of the photoreceptor connecting cilium in the delivery of opsin to forming discs and in the maintenance of membrane domains (Besharse, J. C., and K. H. Pfenninger (1980) J. Cell Biol. 87: 451-463), we have studied developing photoreceptors of neonatal rats during the period of initial disc formation using conventional freeze-fracture, immunocytochemistry, and lectin cytochemistry. Specific anti-opsin-binding sites were localized in the distal cilium, the developing outer segment plasma membrane, and at focal sites on the inner segment plasma membrane at all developmental stages examined, including the period prior to the onset of disc morphogenesis. The proximal ciliary shaft generally lacked anti-opsin-binding sites or exhibited them in extremely low density. The distribution of anti-opsin-binding sites corresponded in a general way to the distribution of large intramembranous particles (IMPs) in freeze-fracture replicas like those seen in the rod outer segment (ROS). The proximal zone corresponded in freeze-fracture images to a zone of consecutive horizontal rows of intramembrane particles (ciliary necklaces) and axoneme-membrane cross-linkers. Although protoplasmic face leaflet IMPs similar to those of the distal cilium and outer segment were less abundant in the inner segment and proximal cilium than in the distal cilium and ROS, they were detected in these zones at low frequency. Cytochemistry with concanavalin A and wheatgerm agglutinin revealed the presence of a well developed glycocalyx in the proximal zone. Although opsin binds both lectins, the results suggest heterogeneity among the glycoconjugates of the three membrane domains. Our data define distinct membrane domains of the developing photoreceptor cilium that have important implications for the mechanisms for delivering and sequestering opsin in the outer segment. They also establish that the mechanism of opsin delivery to the distal zone occurs well in advance of the period of disc morphogenesis.

Animals↗
PubMed1984

Partial characterization of lectin binding sites of retinal photoreceptor outer segments and interphotoreceptor matrix.

We have used cytochemistry together with exoglycosidase digestion and polyacrylamide gel electrophoresis to partially characterize lectin binding sites of the interphotoreceptor matrix and of photoreceptor outer segments. In order to obtain uniform access of reagents to all regions of the preparation, we have used a procedure in which plastic sections are etched with sodium ethoxide prior to cytochemical analysis. Neuraminidase pretreatment of plastic-embedded sections of Xenopus laevis eyecups leads to a loss of wheat germ agglutinin binding and a concomitant appearance of Ricinus communis agglutinin binding to the interphotoreceptor matrix. In contrast, wheat germ agglutinin binding to the outer segments is not altered by the neuraminidase pretreatment. These results suggest that wheat germ agglutinin binding sites of interphotoreceptor matrix are sialoglyconjugates and that outer segment binding sites are not sialoglycoconjugates. Enzyme digestions followed by lectin cytochemistry of matrix polypeptides separated by polyacrylamide gel electrophoresis do not identify a likely candidate to give rise to the cytochemical staining patterns. Lectin cytochemistry of retinas from which the interphotoreceptor matrix has been extracted do not show a loss of wheat germ agglutinin binding sites to the matrix. These results suggest that the major wheat germ agglutinin binding sites in the interphotoreceptor matrix are to as yet unidentified sialoglycoconjugates.

Animals↗
PubMed1984

Regulation and possible role of serotonin N-acetyltransferase in the retina.

The activity of retinal serotonin N-acetyltransferase (NAT) (arylamine acetyltransferase, EC 2.3.1.5), the penultimate enzyme in melatonin biosynthesis, exhibits properties of a circadian rhythm comparable to that seen in the pineal gland. Using an eye cup preparation we have found that circadian properties persist in vitro, which indicates that an endogenous circadian oscillator controlling NAT is present in the eye. Nighttime increases in NAT activity are suppressed by light, protein synthesis inhibitors, and catecholamines. In light, NAT activity is induced by conditions expected to increase intracellular levels of cyclic AMP (cAMP). This suggests that catecholamines and cAMP are normally involved in the regulation of NAT. Circadian indoleamine metabolism may play a role in the control of rhythmic photoreceptor metabolism as evidenced by the observation that melatonin and related compounds are potent activators of disk shedding.

Acetyltransferases↗
PubMed1983

Regulation of indoleamine N-acetyltransferase activity in the retina: effects of light and dark, protein synthesis inhibitors and cyclic nucleotide analogs.

The regulation of indoleamine N-acetyltransferase (NAT) in the posterior eye was investigated in vivo, and in vitro in cultured eye cups. Surgical separation of neural retina from the retinal pigment epithelium-choroid complex indicated that NAT was localized to neural retina. The activity of retinal NAT fluctuated in vivo in a rhythmic fashion, with peak activity in the dark phase of the light-dark cycle. The rhythm of NAT activity persisted for up to 3 days in constant darkness, with a rhythmic period of approximately 25 h. The rhythm was suppressed by constant light, and could be phase-shifted by exposure to a new light-dark cycle. These observations indicate that retinal NAT activity occurs as a circadian rhythm that is entrained by light and dark. Retinas also responded to light and dark in vitro with changes of NAT activity. A significant increase in retinal NAT activity occurred in eye cups cultured in darkness during the dark phase of the light-dark cycle. This increase was completely suppressed in eye cups cultured at the same time of day in light. The dark-induced increase in NAT was completely blocked by protein synthesis inhibitors, and mimicked in light by cyclic AMP analogs. The similarity of the regulation of NAT activity in retina to that in pineal, and the possible relationship of the retinal NAT rhythm to cyclic metabolism in photoreceptors are discussed.

Acetyltransferases↗
PubMed1983

Methoxyindoles and photoreceptor metabolism: activation of rod shedding.

Using an in vitro eye-cup preparation, we have evaluated a potential relationship between methoxyindole metabolism and photoreceptor disk shedding. Melatonin, 6-chloromelatonin, and 5-methoxytryptophol all activate rod disk shedding in culture. The effect is compound specific since serotonin and N-acetylserotonin are without effect, and it is similar to shedding in vivo because it is evoked by light and is quantitatively comparable to a normal intact animal response. The results suggest the involvement of 5-methoxyindoles in the control of rhythmic photoreceptor metabolism.

Animals↗
PubMed1983

Rod photoreceptor disc shedding in eye cups: relationship to bicarbonate and amino acids.

We previously demonstrated that disc shedding in eye cups from Xenopus laevis depends on medium bicarbonate concentration, but also found that other unidentified medium components influenced the response. Here, in order to determine which medium components influence shedding, we used high bicarbonate salt solutions supplemented with medium components in amphibian tissue culture medium. Compared to the high bicarbonate salt solution alone, addition of fetal bovine serum or whole egg ultrafiltrate enhanced disc shedding. However, a response fully comparable to that in intact animals was obtained on addition of a mixture of 14 amino acids. The full light-evoked response depended on the simultaneous presence of high bicarbonate (35 mM) and amino acids. In the presence of amino acids, photoreceptor tips and RPE showed tight interdigitation and less tendency for in vitro separation, suggesting that these medium components promote such interdigitation. Using the defined medium we have additionally shown that shedding is related to HCO3- concentration under conditions of controlled medium pH, Cl- content and osmolality.

Amino Acids↗
PubMed1983

Photoreceptor disc shedding in eye cups. Inhibition by deletion of extracellular divalent cations.

To further define the medium requirements for in vitro rod disc shedding and phagocytosis in eye cups of Xenopus laevis, the effect of deletion of divalent cations was examined. Calcium-free medium completely eliminated both normal diurnal disc shedding (initiated by light onset) and dark-primed disc shedding (initiated by a period of darkness followed by additional darkness or light). The effect was reversible. Furthermore, the events that occurred during the initial dark-priming period did not require extracellular millimolar calcium, since the addition of calcium (1.8 mM) after an initial hour of darkness in calcium-free medium resulted in a marked increase in disc shedding, regardless of the subsequent lighting condition. Magnesium-free medium did not inhibit light-evoked shedding. However, magnesium-free medium partially inhibited disc shedding in one of the two lighting paradigms used to elicit dark-primed disc shedding. This suggests that the extracellular divalent cation requirement varies for different lighting paradigms that promote shedding. The inhibition of disc shedding by magnesium-free medium was morphologically distinct from calcium-free medium; the inhibition in magnesium-free medium was correlated uniquely with a reduction in the interdigitation between the photoreceptor and the retinal pigment epithelium.

Animals↗
PubMed1982

Effects of cyclic adenosine 3',5'-monophosphate on photoreceptor disc shedding and retinomotor movement. Inhibition of rod shedding and stimulation of cone elongation.

As a test of the hypothesis that cyclic nucleotides play a role in the regulation of retinomotor movements and disc shedding in the photoreceptor-pigment epithelial complex, we have used an in vitro eyecup preparation that sustains both disc shedding and cone retinomotor movements, Eyecups were prepared in white light from animals in which both shedding and cone movement had been blocked by 4 d of constant-light treatment. In eyecups incubated for 3 h in light, disc shedding was negligible and cones remained in the light-adapted (contracted) position. In eyecups incubated in darkness, however, a massive shedding response (dominated by rod photoreceptors) was induced, and at the same time cone photoreceptors elongated to their dark-adapted position. In eyecups incubated in light dbcAMP promoted cone elongation and thus mimicked darkness; the dbcAMP effect was potentiated by the phosphodiesterase inhibitors papaverine and 3-isobutylmethylxanthine. In eyecups incubated in darkness, on the other hand, both phosphodiesterase inhibitors and dbcAMP reduced the phagosome content of the pigment epithelium. The effects of dbcAMP on the cone elongation and rod shedding appear to be specific in that dbcGMP, adenosine, and adenosine 5'-monophosphate had no significant effect. Our results suggest that cAMP plays a role in the regulation of both retinomotor movements and disc shedding.

Animals↗
PubMed1980

Membrane assembly in retinal photoreceptors I. Freeze-fracture analysis of cytoplasmic vesicles in relationship to disc assembly.

To study precursor-product relationships between cytoplasmic membranes of the inner segment of photoreceptors and the continually renewed outer disc membrane, we have compared the density and size distribution of intramembrane particles (IMP) in various membrane compartments of freeze-fractured photoreceptor inner and outer segments. Both rod and cone outer segments of Xenopus laevis are characterized by a relatively uniform distribution of approximately 4,400-4,700 IMP/micron2 in P-face (PF) leaflets of disc membranes. A similar distribution of IMP is found in the outer segment plasma membrane, the ciliary plasma membrane, and in the plasma membrane of the inner segment in the immediate periciliary region. In each case the size distribution of IMP can be characterized as unimodal with a mean diameter of approximately 10 nm. PF leaflets of endoplasmic reticulum, Golgi complex, and vesicles near the cilium have IMP with a size distribution like that in the cilium and outer segment, but with an average density of approximately 2,000/micron2. In contrast, IMP are smaller in average size (approximately 7.5 nm) in PF leaflets of inner segment plasma membrane, exclusive of the periciliary rgion. The similarity of size distribution of IMP in inner segment cytoplasmic membranes and those within the plasmalemma of the cilium and outer segment suggest a precursor-product relationship between the two systems. The structure of the vesicle-rich periciliary region and the segregation of IMP with different size distributions in this region suggest that components destined for incorporation into the outer segment exist as preformed membrane packages (vesicles) which fuse with the inner segment plasma membrane in the periciliary region. Subsequently, membrane components may be transferred to forming discs of the outer segment via the ciliary plasma membrane.

Animals↗
PubMed1979

Purification and characterization of branched chain alpha-ketoacid dehydrogenase from bovine liver mitochondria.

Branched chain alpha-ketoacid dehydrogenase (EC 1.2.4.3(4)) was solubilized and purified from bovine liver mitochondria for the first time. Decarboxylation of alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, and alpha-ketoisocaproate was catalyzed by this multienzyme complex and this activity was co-purified for each substrate. Three enzymatic functions were contained in the complex including decarboxylation of the above ketoacids, transacylation of their simple acid derivatives, and reduction of NAD+ as an overall reaction. Product stoichiometry of these three reactions was 1 CO2:1 acyl-CoA:1 NADH. Activity depended upon the addition of thiamin pyrophosphate, CoASH, and NAD+ which were dissociable cofactors. Physically, two active forms of the enzyme complex were found: a 275,000-dalton unit and a 2 x 10(6)-dalton component. Both showed a characteristic flavin spectra and catalyzed all functions of the complex, implying that 10 small units aggregated into the larger unit. The soluble complex as visualized by electron microscopy had a diameter ranging from 12 to 24 nm corresponding to a molecular weight of 2 x 10(6). The size of the native membrane-bound component remains to be determined.

Amino Acids, Branched-Chain↗
PubMed1979

Turnover of mouse photoreceptor outer segments in constant light and darkness.

Pigmented mice differ from frogs in that lighting regime has only a small effect on rod outer segment turnover. During 8 days in constant light or darkness, disc addition measured as total displacement of a radioactive band in rod outer segments of mice which has received injections of tritiated amino acids was never modified by more than 7% compared to cyclic light controls. Disc shedding continued in each lighting regime, and as judged from outer segment dimensions, disc shedding approximately balanced disc addition.

Animals↗
PubMed1977

Photoreceptor outer segments: accelerated membrane renewal in rods after exposure to light.

The rate of rod outer segment renewal in Rana pipiens tadpoles under constant light and under diurnal conditions of 12 or 2 hours light per day is significantly increased compared to that in animals in darkness. Furthermore, during 24 hours in light after 6 days in darkness the rate of renewal is three to four times that in darkness. In Xenopus laevis tadpoles the rate of renewal is more than five times greater during the first 8 hours of a normal diurnal cycle than during the following 16 hours. These observations demonstrate that bursts of renewal activity occur as a response to light, and suggest that a normal pattern of light alternating with darness plays a fundamental role in the regulation of rod outer segment turnover.

Animals↗
PubMed1977

Turnover of rod photoreceptor outer segments. I. Membrane addition and loss in relationship to temperature.

Membrane turnover in outer segments of Rana pipiens red rods (ROS) was studied in tadpoles maintained under cyclic lighting (12L:12D) at 23 degrees, 28 degrees, and 33 degrees C. Large fragments (greater than 2 microns in diameter or length) were shed from the ROS tips shortly after the onset of light. These were phagocytized by the pigment epithelium (PE) which caused an increase in the number of phagosomes greater than 2 microns in size (large phagosomes). Large phagosomes were present in highest numbers 2-4 h after light exposure and were degraded by 8-12 h. The proportion of ROS that shed each day after the onset of the light cycle increased with increment increases in temperatures (23 degrees C-18%, 28 degrees C-33%, 33 degrees C-42% per day), resulting, in a reduction in the average interval of time between repeated sheddings (23 degrees C-5.6 days, 28 degrees C-3 days, 33 degrees C-2.4 days) though the average numbers of disks shed from ROS at the various temperatures were not significantly different (23 degrees C-139.5 +/- 5.7, 28 degrees C-129.4 +/- 7.6, 33 degrees C-129.9 +/- 4.8 disks/shed packet). Phagosomes in the PE that were less than 2 microns in diameter (small phagosomes) were present in relatively constant numbers throughout the day, and their numbers increased at higher temperatures. The absence of a concomitant increase in small phagosomes as large phagosomes were degraded indicates that large phagosomes were not the major source of small phagosomes. When the PE was isolated to culture in the absence of the retina, these small phagosomes were degraded. The rate of disk addition to the ROS base was determined by autoradiography after [3H]leucine injection. The number of disks added per day increased with elevations of temperature (23 degrees C-32.4; 28 degrees C-55.9; 33 degrees C-65.5). The average number of disks added to the ROS between repeated sheddings (23 degrees C-181.4; 28 degrees C-167.7; 33 degrees C-157.2) was greater than the number of disks shed after light exposure. Inasmuch as the ROS show no net increase in length during the tadpole stages utilized, the remaining disks must be lost at some other time. Electron microscope analysis revealed the presence of small groups of disks in curled configurations at the tips of ROS, suggesting possible stages of detachment.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1977

Turnover of rod photoreceptor outer segments. II. Membrane addition and loss in relationship to light.

The rate of disk addition to rod outer segments (ROS) varies widely in Xenopus laevis tadpoles kept in cyclic light (12L:12D). When measured as radioactive band (3H-band) displacement during the 2nd day after injection of [3H]leucine, 75% of the daily increment of displacement occurred during the first 8 h of light. During the same interval, the number of open disks at the ROS base increased more than threefold. During the last 8 h of darkness, 3H-band displacement was undetectable and the number of open disks was reduced. These observations suggest the possibility that disk addition may occur discontinuously. During the 3rd and 4th days after injection of [3H]leucine, maximal displacement of the 3H-band occurred later in the day than on the 2nd day, its movement no longer corresponding to the increase in open disks. This delay in 3H-band displacement may reflect a time delay as a result of propagation of compressive stress in an elastic ROS system. Maximal disk loss from ROS as reflected in counts of phagosomes in the pigment epithelium occurred within 1 h of light exposure, and phagosome counts remained high for 4 h before declining to a low level in darkness. Modified lighting regimes affected the daily rhythms of shedding and disk addition differently, suggesting that control mechanisms for the two processes are not directly coupled. During 3 days in darkness, disk addition was reduced 50% compared to controls (12L:12D), whereas shedding was reduced by about 40%. Although reduced in level, shedding occurred as a free-running circadian rhythm. There was no evidence of rhythmicity of disk addition in darkness. In constant light, the rate of disk addition was not different from controls, but shedding was reduced by about 80% after the 1st day. This resulted in a 21% increase in ROS length. Among animals kept on a 2.5L:21.5D cycle, the rate of disk addition was reduced by 40% while shedding was maintained near control levels, resulting in a slight decrease in ROS length. These observations indicate that normal shedding requires alternating light and darkness, and that the daily rhythm of disk addition is due primarily to daily stimulation by light.

Animals↗