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.     

Evidence for melanogenesis in the retinal pigment epithelium of adult cattle and golden hamster.

1. The ultrastructure of the retinal pigment epithelium (RPE) of adult Syrian golden hamsters and cattle was examined with respect to pigment granules and phagosomes involved in degradation of disk membranes from rod outer segments. 2. In the RPE of cattle, phagosomes were found that contained an electron-dense melanin-like material that was not autofluorescent and therefore not lipofuscin. 3. Disk membranes of rods are about 4 nm thick and become enlarged (7-20 nm) and electron-dense during degradation in the RPE. 4. Additionally electron-dense vesiculo-globular bodies (10-100 nm) were found in phagosomes during disk membrane degradation and in mature melanin granules. 5. In the RPE of adult hamsters that had been exposed to intense light, premelanosomes containing unmelanised filaments with a striated periodicity were found in the cytoplasm or in association with mature melanin granules. Early and late stage melanosomes were also present. Phagosomes in the RPE contained degraded disk membranes, melanin-like material and melanofilaments. 6. Dopa oxidase was detected ultrastructurally within shed disk membranes that were in close contact with the microvilli of the RPE. 7. The possibility of melanogenesis within phagosomes during disk membrane degradation is discussed.     

The molecular regulation of organelle transport in mammalian retinal pigment epithelial cells

Retinal pigment epithelial cells contain large numbers of melanosomes that can enter the apical processes extending between the outer segments of the overlying photoreceptors. Every day the distal portion of the photoreceptor outer segment is shed and phagocytosed by the retinal pigment epithelial cell. The phagosome is then transported into the cell body and the contents degraded by lysosomal enzymes. This review focuses on recent progress made in the identification of molecules that regulate the transport of melanosomes into the apical processes and the transport of phagosomes into the cell body. Myosin VIIa is a key player in both processes and, at least in the case of melanosome movement, myosin VIIa is recruited to the melanosome via the GTPase, Rab27a. The possible role played by defects in the transport of melanosomes and phagosomes in the development of retinal degenerative diseases is discussed.     

Immunocytochemical localization of opsin in outer segments and Golgi zones of frog photoreceptor cells. An electron microscope analysis of cross-linked albumin-embedded retinas

Adult vertebrate retinal cells (rod and cones) continuously synthesize membrane proteins and transport them to the organelle specialized for photon capture, the outer segment. The cell structures involved in the synthesis of opsin have been identified by means of immunocytochemistry at the electron microscope level. Two indirect detection systems were used: (a) rabbit antibodies to frog opsin were localized with ferritin conjugated F(ab')2 of sheep antibodies to rabbit F(ab')2 and (b) sheep antibodies to cattle opsin were coupled to biotin and visualized by means of avidin-ferritin conjugates (AvF). The reagents were applied directly to the surface of thin sections of frog retinal tissues embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Specific binding of anti-opsin antibodies indicates that opsin is localized in the disks of rod outer segments (ROS), as expected, and in the Golgi zone of the rod cell inner segments. In addition, we observed quantitatively different labeling patterns of outer segments of rods and cones with each of the sera employed. These reactions may indicate immunological homology of rod and cone photopigments. Because these quantitiative variations of labeling density extend along the entire length of the outer segment, they also serve to identify the cell which has shed its disks into adjacent pigment ipithelial cell phagosomes.     

Production of Sarcocystis cruzi sporocysts by dogs fed experimentally infected and naturally infected beef.

Twenty-one coccidia-free dogs were fed either experimentally infected or naturally infected beef in 4 experiments. The pretreatment period ranged from days 9 to 33; 7 dogs shed their first sporocysts on day 12. The full length of the patent period was not determined because most dogs were still shedding sporocysts when experiments ended on days 40 and 60. Although the patent period was long, sporocysts were not shed continuously; the total number of days sporocysts were actually shed ranged from 3 to 40. The average number of sporocysts shed by dogs fed 454 to 908 g of beef ranged from 861,000 to over 20 million. Most sporocysts were shed from days 15 to 30; peak numbers were shed on days 23 and 24. Average peak numbers ranged from 145,000 to 408,400 sporocysts. Such data can be applied to facilitate collection of sporocysts in the laboratory or to estimate the potential for transmission of infective organisms under field conditions.     

Effects of high temperature on body temperature and hormonal adjustments in piglets

Effects of a high (33 degrees C) or thermoneutral (23 degrees C) temperature on body temperature and endocrine parameters were studied in weaned piglets. Rectal and skin temperatures were measured in four ad libitum fed animals per temperature during three weeks. After this acclimation period, 11 blood samples were withdrawn on a 24-h period. Over the acclimation period, rectal and skin temperatures were 0.6 and 2.9 degrees C higher, respectively, at 33 degrees C than at 23 degrees C (P < 0.01), this change occurring from the 1st day at 23 or 33 degrees C. A tendency of serum leptin concentrations to be lower after meals at 33 degrees C than at 23 degrees C was also displayed (P = 0.09). Plasmatic concentrations in Insulin-like growth factor I and thyroxine were decreased at 33 degrees C relative to 23 degrees C (P < 0.01 and P < 0.06, respectively), and triiodothyronine concentrations tended to be lower at 33 degrees C than at 23 degrees C (P = 0.1), which could account for the lower heat production and growth observed in pigs exposed to high temperatures.     

Effect of storage time and temperature and the variation among replicate tests (on different days) on the performance of spore disks and strips.

Laboratory-prepared spore disks were stored for 96 weeks at 22 degrees C with 50% relative humidity (RH) and at 4 degrees C with less than 1% RH. At the same time commercial spore strips were stored for 64 weeks at 22 degrees C with 50% RH. The spore count per unit and the heat resistance were measured at the beginning of the experiment and after 16, 32, 48, 64, 80, and 96 weeks of storage. The laboratory-prepared spore disks stored at 4 degrees C with less than 1% RH showed less change in numbers of spores per disks and decrease in the survival time than did the disks stored at 22 degrees C with 50% RH. Both the laboratory-prepared spore disks and the commercial spore strips stored at 22 degrees C with 50% RH decreased in survival times with increased storage time. The relative change in the survival times with storage was less for the commercial spore strips than for the laboratory-prepared spore disks.     

Reactive oxygen species in pregnant rats: effects of exercise and thermal stress

With the aim of evaluating the effect of interaction between physical training or exercise only during pregnancy and thermal stress on oxidative stress, and antioxidant mechanism sedentary pregnant rats (PS), exercised pregnant rats only during pregnancy (PE) and trained rats submitted to also exercise during pregnancy (PT) were compared (N=63). Exercise sessions consisted of swimming at 80% of maximal work load supported into water at 28 degrees C (hypothermia, PS 28, PE28, PT28) or 35 degrees C (thermal neutrality, PS35, PE35, PT35) or 39 degrees C (hyperthermia, PS39, PE39, PT39), for 30 min. The initial body weight in all groups of rats was from 177 to 207 g. On the 20th day of pregnancy, 24 h after the last immersion or swimming session venous blood was collected to determine oxidative stress. Plasma concentrations of means malondialdehyde (MDA) values measured as thiobarbituric acid reactive substances (TBARS); total glutathione (GSH) and vitamin E were determined. The oxidative stress index was calculated from the ratio TBARS/GSH and TBARS/Vitamin E. TBARS did not change on the group PE at different temperatures of water; TBARS were higher for PS28 than PS35 and PS39; PT35 had higher values than PT28 and PT39. For GSH, PS39 was lower than PS35; PE28 was higher than PE35 and PE39 and PT35 were lower than PT28 and PT39. Plasma concentration of vitamin E did not present any difference for sedentary rats at different water temperatures, but for PE28, the values were lower than for PE35 and PE39, whereas PT39 was lower than PT35 and PT28. In relation to TBARS/GSH, it was verified an increase in oxidative stress for PS28 (in relation to PS35 and PS39), PE35, and PT35 (in relation to PE28 and PE39 or PT28 and PT39); regarding the ratio TBARS/vitamin E, the highest values were obtained at 35 degrees C for PS and PT groups and at 39 for PE group. These results have shown the great complexity of the interaction between physical training, thermal stress and pregnancy. Apparently, hypothermia produces large index of oxidative stress only in sedentary rats, but this index was greater at 35 degrees C in relation to extreme temperatures for trained rats. These results have suggested that physical training allows a more efficient activation of antioxidant mechanisms under thermal stress.     

Effects of mutations in apolipoprotein C-1 on the reconstitution and kinetic stability of discoidal lipoproteins

To probe the role of protein conformation in the formation and kinetic stability of discoidal lipoproteins, thermal unfolding and refolding studies were carried out using model lipoproteins reconstituted from dimyristoylphosphatidylcholine (DMPC) and selected mutants of human apolipoprotein C-1 (apoC-1). Circular dichroism (CD) spectroscopy and electron microscopy show that the Q31P mutant, which has alpha-helical content in solution (33%) and on DMPC disks (67%) similar to that of the wild type (WT), forms disks of smaller diameter, = 13 nm, compared to 17 nm of the WT-DMPC disks. The L34P mutant, which is largely unfolded in solution, forms disks with alpha-helix content and diameter similar to those of the WT. The R23P mutant, which is fully unfolded in solution, forms disks that have similar diameter but reduced alpha-helix content (40%) compared to the WT-DMPC disks (65%). Remarkably, despite large variations in the alpha-helix content or the disk diameter among different mutant-DMPC complexes, the mutations have no significant effect on the unfolding rates or the Arrhenius activation energy of the disk denaturation, E(a) = 25-29 kcal/mol. This suggests that the kinetic stability of the discoidal complexes is dominated by the lipid-lipid rather than the protein-lipid interactions. In contrast to the heat denaturation, the lipoprotein reconstitution upon cooling monitored by CD and light scattering is significantly affected by mutations, with Q31P forming disks in the broadest and R23P in the narrowest temperature range. Our results suggest that the apolipoprotein helical structure in solution facilitates reconstitution of discoidal lipoproteins but has no significant effect on their kinetic stability.     

残次油松林群落特征与生物多样性恢复

在河北省兴隆县雾灵山国家级自然保护区研究了经长期人为破坏的残次油松林的群落特征及其在不同保护年限下的生物多样性恢复规律。研究表明,与正常林分相比,残次林的物种多样性指数大幅度降低,物种数量显著减少。在植物和昆早群落各参数的变化中,以灌木和天敌昆虫类群受影响最大,其科数分别减少５５．５６％和６０％,科－种多样性指数分别减少３０．１％和６０．７２％。     

Post-natal development of brown adipose tissue in the rat bred at 23 degrees C or 28 degrees C.

In view to study the effects of thermal environment on the development and the thermogenic activity of interscapular brown adipose tissue (BAT), young rats born at 23 degrees C or 28 degrees C were sacrificed at 1, 3, 7, 11, 14 or 21 days after birth. The rate of increase in animal weight was quite the same at both temperatures up to the 14th day. The development of BAT and its contents in lipids, in water and in noradrenaline indicate that the energetic activity of the tissue is greatly stimulated in rats kept at 23 degrees C up to the 11th day. It is concluded that in rats bred in the habitual thermal conditions (23 degrees C), the occurrence of non shivering thermogenesis (NST) is important during the period of ten days after birth; in the following period NST could be progressively replaced by other thermoregulatory processes.     

Phycobilisome composition and possible relationship to reaction centers

The photosynthetic apparatus was studied in Anacystis nidulans wild type and in a spontaneous pigment mutant 85Y which had improved growth in far-red light (greater than 650 nm). Two phycobiliproteins, C-phycocyanin (lambda max 625) and allophycocyanin (lambda max 650), were present in a molar ratio of approximately 3:1 in the wild type and approximately 0.4:1 in the mutant. Phycobilisomes of wild type cells were larger (57 X 30 nm) than those of the mutant 85Y (28 X 15 nm). In the mutant they seemed to consist primarily of the allophycocyanin core. Fluorescence emission maxima of wild type and mutant 85Y phycobilisomes were at 680 nm (23 degrees C) and 685 nm (-196 degrees C). Excitation maxima of phycobilisomes were at 630 and 650 nm for the wild type and the mutant 85Y, respectively. The phycobilisomes of wild type cells whether grown in white or far-red light had the same size and pigment composition. A typical wild type cell in white light had a thylakoid area of 22.8 microns 2, but in far-red light the area was reduced to 13.5 microns 2, which was close to that of 85Y at 13.6 microns 2. Chlorophyll molecules per cell decreased in far-red light from 1.1 X 10(7) in wild type (white light) to 4.5 X 10(6) in mutant 85Y (far-red). The number of phycobilisomes per cell (approx 2 X 10(4)), calculated from the phycobiliprotein content and phycobilisome size, was about the same in wild type (white light) and mutant 85Y (far-red light), but the number of phycobilisomes per unit area of thylakoid was significantly greater in mutant 85Y than in wild type. The present results suggest that the phycobilisomes are linked with reaction centers and that the PSII complement (photo-system II and phycobilisome) was fully maintained in far-red light.     

Linkage of Retinal to Opsin

RHODOPSIN, the visual pigment of vertebrate rods, has been shown to consist of a chromophore (11-cis retinal) bound to a protein (opsin)^1–2. It has been proposed that the linkage is a Schiff base between phosphatidyl ethanolaniine (PE) and retinal and that when exposed to light, the retinal migrates from PE to the ε-amino-group of a lysine residue in opsin^3–7. Most of the support for this theory comes from the observation that N-retinylidenephosphatidylethanolamine (N-RPE) can be extracted in the dark from rod outer segments (ROS)^3,4. Furthermore, N-retinylphosphatidylethanolamine (N-RH₂PE) has been extracted from ROS preparations after treating the visual pigment with acid and NaBH₄—conditions which are assumed fix retinal to its “native” binding site through a secondary amine linkage⁷. All these studies, however, were carried out on crude extracts of ROS in various detergents. These crude extracts contain large amounts of phospholipid and retinal which is not bound to opsin. Thus, the isolation of N-RPE from crude ROS extracts does not necessarily point to its involvement in the binding of retinal to opsin. In contradiction to these reports are findings that purified visual pigment contains no phospholipid^9,10 and that the molar concentration of N-RPE in bovine ROS is less than that of rhodopsin¹¹. We have taken advantage of the observation that visual pigment in the outer segment disks is continually being renewed¹² to label the rhodopsin with ³H-retinal and to show in yet another way that N-RPE does not exist in purified visual pigment.     

Changes in the composition of the lysosomes in hepatocytes cultured in vitro in modelling pathological states

The monolayer cultures of newborn rats hepatocytes were treated with substrate starvation (20 min.) anoxia accompanied with substrate starvation (1 h.), following rehabilitation (1 h.) and cooling (4 degrees C, 30 min). After the neutral red staining we have examined the alterations in lysosomes quantity and the development of phagocytosis; we also tested the hepatocytes distribution due to the amount of the second lysosomes per cell. We have shown that a short time pathological treatment caused a decrease in large lysosomes (1-3 microns) amount in significant portion of hepatocytes. There were neither increase in phagosomes amount per cell nor alterations in the quantity of cells, containing phagosomes. However a long time pathological treatment caused the increase in phagosomes and cells. We propose that initial stage of lysosomal apparatus alterations, connected with reduction of CAMP level, is accompanied by a stamping of large lysosomes.     

Strength of Ca(2+) binding to retinal lipid membranes: consequences for lipid organization

There is evidence that membranes of rod outer segment (ROS) disks are a high-affinity Ca(2+) binding site. We were interested to see if the high occurrence of sixfold unsaturated docosahexaenoic acid in ROS lipids influences Ca(2+)-membrane interaction. Ca(2+) binding to polyunsaturated model membranes that mimic the lipid composition of ROS was studied by microelectrophoresis and (2)H NMR. Ca(2+) association constants of polyunsaturated membranes were found to be a factor of approximately 2 smaller than constants of monounsaturated membranes. Furthermore, strength of Ca(2+) binding to monounsaturated membranes increased with the addition of cholesterol, while binding to polyunsaturated lipids was unaffected. The data suggest that the lipid phosphate groups of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in PC/PE/PS (4:4:1, mol/mol) are primary targets for Ca(2+). Negatively charged serine in PS controls Ca (2+) binding by lowering the electric surface potential and elevating cation concentration at the membrane/water interface. The influence of hydrocarbon chain unsaturation on Ca(2+) binding is secondary compared to membrane PS content. Order parameter analysis of individual lipids in the mixture revealed that Ca(2+) ions did not trigger lateral phase separation of lipid species as long as all lipids remained liquid-crystalline. However, depending on temperature and hydrocarbon chain unsaturation, the lipid with the highest chain melting temperature converted to the gel state, as observed for the monounsaturated phosphatidylethanolamine (PE) in PC/PE/PS (4:4:1, mol/mol) at 25 degrees C.     

Processing of ribosomal RNA in a temperature sensitive mutant of BHK cells.

The processing of ribosomal RNA has been studied in a temperature sensitive mutant of the Syrian hamster cell line BHK 21. At 39 degrees C, these cells are unable to synthesize 28S RNA, and 60S ribosomal subunits, while 18S RNA, and 40S subunits are produced at both temperatures. At 39 degrees C the 45S RNA precursor is transcribed and processed as in wild type cells. The processing of the RNA precursors becomes defective after the cleavage of the 41S RNA, and the separation of the 18S and 28S RNAs sequences in two different RNA molecules. The 36S RNA precursor, which is always present in very small quantity in the nucleoli of wild type cells and of the mutant at 33 degrees C, is found in very large amounts in the mutant at 39 degrees C. The 36S RNA can be, however, slowly processed to 32S RNA. The 32S RNA cannot be processed at 39 degrees C, and it is degraded soon after its formation. Only a small proportion accumulates in the nucleoli. The 32S RNA synthesized at 39 degrees C cannot be processed to 28S RNA upon shift to the permissive temperature, even when the processing of the newly synthesized rRNA has returned to normal. The data suggest that the 36S and 32S RNAs are contained in aberrant ribonucleoprotein particles, leading to a defective processing of the particles as a whole.     

Pigment epithelial ensheathment and phagocytosis of extrafoveal cones in human retina.

The association between extrafoveal cone outer segments and pigment epithelial cells was studied by transmission electron microscopy in three human retinas; ages 5,45 and 60. The pigment epithelial apical surface from a fourth human retina, age 38,was viewed in the scanning electron microscope. Multiple villous-like apical processes protrude from the pigment epithelium into the space above each cone. Sometimes one or more of these processes is sheet-like in form and contains a wealth of intracellular organelles, including mitochondria. One or more of the villous-like procesess reaches the cone and expands to ensheath the upper one-third of the outer segment. Llike vertebrate rods, extrafoveal human cones shed their terminal disks in packets and these packets are phagocytosed by the ensheathing apical processes. The phagosomes then ascend in the processes toward the pigment epithelia soma. Digestion of phagosomes appears to begin in the apical processes.     

镰形臂尾轮虫和尾突臂尾轮虫的生活史特征比较

应用单个体培养方法,以浓度为3.0×10⁶ cells·ml^-1的斜生栅藻为食物,在18 ℃、23 ℃、28 ℃和33 ℃的温度梯度下比较了镰形臂尾轮虫和尾突尾轮虫的生活史特征.结果表明：18 ℃和23 ℃下,尾突臂尾轮虫的生殖期和平均寿命均显著长于镰形臂尾轮虫,产卵量也显著大于镰形臂尾轮虫;28 ℃下,2种轮虫的各主要发育阶段历时、平均寿命和产卵量均无显著差异;33 ℃下,镰形臂尾轮虫的生殖期和生殖后期历时以及平均寿命均显著长于尾突臂尾轮虫,产卵量显著大于尾突臂尾轮虫.18 ℃下,尾突臂尾轮虫的生命期望、净生殖率和种群内禀增长率均显著大于镰形臂尾轮虫;23 ℃和28 ℃下,尾突臂尾轮虫的生命期望显著长于镰形臂尾轮虫,而其他种群增长参数间均无显著差异;33 ℃下,镰形臂尾轮虫的世代时间、生命期望、净生殖率和种群内禀增长率均极显著大于尾突臂尾轮虫.2种轮虫的主要发育阶段历时、平均寿命、产卵量、世代时间、生命期望、净生殖率和种群内禀增长率对温度变化的反应也存在差异.研究结果表明,尾突臂尾轮虫更能适应较低的环境温度,而镰形臂尾轮虫则相反.     

The effect of temperature on the motor activity of the chick embryo and amnion at 5-14 days of development]

It has been shown that cooling the developing eggs from 37.7 degrees C results cessation of motor activity of the amnion in 5-14-day embryos at 36-33 degrees C, whereas motor activity of the embryo remains unaffected up to 31-26 degrees C. Immobilization of the embryo was observed on cooling up to 22-18 degrees C. The recovery of motor activity after cooling during heating takes place in a reverse order. Embryonic movements are observed at 18-23 degrees C, contractions of the amnion--at 28-33 degrees C. These experiments reveal complete independence of embryonic movements from the amnion. Motor activity of the amnion is related to that of the embryo only between the 8th and the 10th day of incubation.     

Storage effects on genomic DNA in rolled and mature coca leaves

Rolled and mature leaf tissue was harvested from Erythroxylum coca var. coca Lam. (coca) to determine a method for storage that would maintain DNA with high quality and content up to 50 days. Harvesting coca leaf tissue under Andean field conditions often requires storage from 3 to 10 days before extraction where tissue integrity is lost. All samples of rolled and mature coca leaf tissue were harvested and separately stored fresh in RNAlater for 50 days at 4 degrees, -20 degrees, and 23 degrees C, while similar samples were air-dried for 72 h at 23 degrees C or oven-dried for 72 h at 40 degrees C after storage, before extraction. Triplicate samples of each tissue type were extracted for DNA at 10-day intervals and showed that DNA integrity and content were preserved in leaf tissue stored at 4 degrees and -20 degrees C for 50 days. Rolled and mature leaf tissue stored at 4 degrees, -20 degrees, and 23 degrees C showed insignificant degradation of DNA after 10 days, and by day 50, only leaf tissue stored at 4 degrees and -20 degrees C had not significantly degraded. All air- and oven-dried leaf tissue extracts showed degradation upon drying (day 0) and continuous degradation up to day 50, despite storage conditions. Amplified fragment length polymorphism analysis of DNA from rolled and mature leaf tissue of coca stored at 4 degrees and -20 degrees C for 0, 10, and 50 days showed that DNA integrity and content were preserved. We recommend that freshly harvested rolled or mature coca leaf tissue be stored at 4 degrees, -20 degrees, and 23 degrees C for 10 days after harvest, and if a longer storage is required, then store at 4 degrees or -20 degrees C.