THE RENEWAL OF PHOTORECEPTOR CELL OUTER SEGMENTS

The utilization of methionine-³H by retinal photoreceptor cells has been studied by radioautographic technique in the rat, mouse, and frog. In all three species, the labeled amino acid is concentrated initially in the inner segment of the cell. Within 24 hr, the radioactive material is displaced to the base of the outer segment, where it accumulates as a distinct reaction band. The reaction band then gradually moves along the outer segment and ultimately disappears at the apex of the cell, which is in contact with the retinal pigment epithelium. These findings are interpreted to indicate that the photoreceptor cell outer segment is continually renewed, by the repeated lamellar apposition of material (membranous discs) at the base of the outer segment, in conjunction with a balanced removal of material at its apex. The outer segment renewal rate is accelerated in frogs when ambient temperature is raised, and is elevated in both frogs and rats when the intensity of retinal illumination is increased.     

THE RENEWAL OF ROD AND CONE OUTER SEGMENTS IN THE RHESUS MONKEY

The renewal of retinal rod and cone outer segments has been studied by radioautography in rhesus monkeys examined 2 and 4 days after injection of leucine-³H. The cell outer segment consists of a stack of photosensitive, membranous discs. In both rods and cones some of the newly formed (radioactive) protein became distributed throughout the outer segment. Furthermore, in rods (but not in cones), there was a transverse band of concentrated radioactive protein slightly above the outer segment base 2 days after injection. This was due to the formation of new discs, into which labeled protein had been incorporated. At 4 days, these radioactive discs were located farther from the outer segment base. Repeated assembly of new discs had displaced them away from the basal assembly site and along the outer segment. Measurements of the displacement rate indicated that each retinal rod produces 80–90 discs per day, and that the entire complement of outer segment discs is replaced every 9–13 days. To compensate for the continual formation of new discs, groups of old discs are intermittently shed from the apical end of the cell and phagocytized by the pigment epithelium. Each pigment epithelial cell engulfs and destroys about 2000–4000 rod outer segment discs daily. The similarity between visual cells in the rhesus monkey and those in man suggests that the same renewal processes occur in the human retina.     

MODULATION OF FATTY ACIDS IN THE MEMBRANES OF ANACYSTIS NIDULANS (CYANOBACTERIA): INCORPORATION OF ODD-NUMBERED CARBON FATTY ACIDS1

Anacystis nidulans Richt., a unicellular cyanobacterium, can incorporate exogenously supplied fatty acids, including odd-numbered carbon fatty acid (OFAs), into the acylglycerols of cell membranes. Data are presented for the uptake of undecanoic acid (11:0) into cells of A. nidulans, the subsequent elongation up to C17, and incorporation of OFA into the four major membrane acylglycerols. The incorporation of OFAs was followed by desaturation of part of the saturated fatty acid to monoenoic fatty acid. Positional analyses of the double bonds of these manoenoic fatty acids suggest that there is one desaturase that inserts a Δ⁹ bond in both odd- and even-numbered fatty acids of varying chain length. Our data also suggest that there is no positional specificity for chain length on the glycerol backbone by the acyltransferases.     

夜行壁虎视细胞外段的更新:[~3H]亮氨酸的渗入和分布

本文报道用光镜放射自显影方法观察夜行壁虎网膜感光细胞外段的更新。当注射氚标记的亮氨酸后,这种由其祖先视锥演变来的夜行壁虎视杆(并且至今仍保留着一些形态学上属视锥的特征)其外段呈现的标记图型仍与一般的视杆细胞相似。     

EFFECTS OF DIFFERENT DIETARY LEVELS OF ESSENTIAL FATTY ACIDS ON THE FATTY ACID COMPOSITION OF ETHANOLAMINE PHOSPHOGLYCERIDES IN MYELIN AND SYNAPTOSOMAL PLASMA MEMBRANES

Abstract— Three dietary levels of essential fatty acids (EFA), 3 0, 0 75 and 0 07 calorie-% were fed to rats for two generations or more. Myelin was isolated at the ages of 18, 30, 45 and 120 days and synaptosomal plasma membranes at 18, 30 and 45 days. No difference was found in the lipid composition between the dietary groups in either subcellular fraction. The fatty acid patterns of ethanolamine phosphoglycerides (EPG) were analysed. In myelin the proportions of 18:1 and 20:1 increased with age, while those of 20:4 (n-6) and 22:6 (n-3) decreased, in synaptosomal plasma membranes the proportions of 20:4 (n-6) decreased with age, but 22:6 (n-3) increased and the sum of the polyunsaturated fatty acids was constant. At no age were significant differences found between the proportions of saturated and monounsaturated fatty acids, in either myelin or the synaptosomal plasma membrane fraction, when the different dietary groups were compared. In myelin from rats fed 007 calorie-% EFA the proportions of 20:4 (n-6) were slightly lower than in the two other groups, while those of 22 6 (n-3) were considerably lower. The synaptosomal plasma membranes fraction of rats fed O-07 calorie-% EFA had equal or slightly larger amounts of 20:4 (n-6) than in the two other groups, while 22:6 (n-3) was considerably smaller. In both subcellular fractions the decreased proportion of fatty acids of linoleic and linolenic acid series was compensated for by an increase in 20:3 (n-9) and 22:3 (n-9). The sum of these two fatty acids was equal in the EPG of myelin and synaptosomal plasma membranes at 18 days of age. At 30 and 45 days of age a lower value was found in the synaptosomal plasma membranes, while in the myelin fraction a slight decrease was found only at 120 days of age.     

KINETICS OF ROD OUTER SEGMENT RENEWAL IN THE DEVELOPING MOUSE RETINA

The kinetics of rod outer segment renewal in the developing retina have been investigated in C57BL/6J mice. Litters of mice were injected with [³H]amino acids at various ages and killed at progressively later time intervals. Plastic 1.5 µm sections of retina were studied by light microscope autoradiography. The rate of outer segment disk synthesis, as judged by labeled disk displacement away from the site of synthesis, is slightly greater than the adult level at 11–13 days of age; it rises to more than 1.6 times the adult rate between days 13 and 17, after which it falls to the adult level at 21–25 days. The rate of disk disposal, as measured by labeled disk movement toward the site of disposal, is less than 15% of the adult level at 11–13 days of age; it rises sharply to almost 70% of the adult level by days 13–15 and then more gradually approaches the adult rate. The net difference in rates of synthesis and disposal accounts for the rapid elongation of rod outer segments in the mouse between days 11 and 17 and the subsequent, more gradual elongation to the adult equilibrium length reached between days 19 and 25. The changing rate of outer segment disk synthesis characterizes the late stages of cytodifferentiation of the rod photoreceptor cells.     

ACTIVATION OF GUANYLATE CYCLASE IN SYNAPTIC PLASMA MEMBRANES OF CEREBRAL CORTEX BY FREE FATTY ACIDS

Guanylate cyclase (EC 4.6.1.2) of synaptic plasma membranes of rat cerebral cortex was stimulated about 6-fold by several unsaturated fatty acids (arachidonic, linolenic, linoleic, oleic, palmitoleic and myristoleic acid). Ricinoleic acid (12-hydroxyoleic acid) was much less effective. Saturated fatty acids (C₁₀ and C₁₄-C₂₀) and the methylester of linoleic acid were ineffective. Stimulation by linoleic acid was influenced by the concentration of enzyme protein. At 480 μg/ml of protein 0.6 mm -linoleic acid produced maximal activation of 6-fold_ Activity stimulated by linoleic acid examined with 1.0 mm -GTP was maximal at pH 7.8-7.9 and with 2 mm -MnCl₂, whereas basal activity showed broad optimal pH and Mn²⁺-concentration dependence. Activation of the enzyme by linoleic acid was only partially reversed by washing. Particulate guanylate cyclase of heart, small intestine, adrenal medulla, liver and lung was also activated by linoleic acid. The extents of activation (1.5-14.7-fold) by linoleic acid and the concentrations (0.2-1.0 mat) required for maximal activation depended on the tissues.     

PARTICIPATION OF THE RETINAL PIGMENT EPITHELIUM IN THE ROD OUTER SEGMENT RENEWAL PROCESS

The disposal phase of the retinal rod outer segment renewal process has been studied by radioautography in adult frogs injected with tritiated amino acids. Shortly after injection, newly formed radioactive protein is incorporated into disc membranes which are assembled at the base of the rod outer segment. During the following 2 months, these labeled discs are progressively displaced along the outer segment owing to the repeated formation of newer discs. When the labeled membranes reach the end of the outer segment, they are detached from it. They subsequently may be identified in inclusion bodies within the pigment epithelium by virtue of their content of radioactivity. These inclusions have been termed phagosomes. Disc membrane formation is a continuous process, but the detachment of groups of discs occurs intermittently. The detached outer segment fragments become deformed, compacted, undergo chemical changes, and are displaced within the pigment epithelium. Ultimately, the material contained in the phagosomes is eliminated from the cell. In this manner the pigment epithelium participates actively in the disposal phase of the rod outer segment renewal process.     

S-ADENOSYLMETHIONINE IN THE BIOSYNTHESIS OF BACTERIAL FATTY ACIDS.

O'Leary, William M. (Cornell University Medical College, New York, N.Y.). S-adenosylmethionine in the biosynthesis of bacterial fatty acids. J. Bacteriol. 84:967-972. 1962.-The fatty-acid contents of a wild strain of Aerobacter aerogenes and of three auxotrophic mutants were determined by the use of gas-liquid chromatography. All strains studied exhibited essentially identical fatty-acid spectra. This organism was shown to contain large amounts of a C(17) cyclopropane fatty acid, and it is suggested that it may be a convenient natural source of this compound. By use of the auxotrophic mutants which have varying requirements for exogenous sources of methionine and S-adenosylmethionine, it was shown by means of radioisotope tracer techniques that S-adenosylmethionine functions in this organism as a donor of a one-carbon unit that is used to form the ring structure in the biosynthesis of cyclopropane fatty acids.     

FATTY ACIDS AND HYDROXY FATTY ACIDS IN THREE SPECIES OF FRESHWATER EUSTIGMATOPHYTES

The monocarboxylic fatty acids and hydroxy fatty acids of three species of freshwater microalgae—Vischeria punctata Vischer, Vischeria helvetica (Vischer et Pascher) Taylor, and Eustigmatos vischeri (Hulbert) Taylor, all from the class Eustigmatophyceae— were examined. Each species displayed a very similar distribution of fatty acids, the most abundant of which were 20:5n-3, 16:0, and 16:1n-7; C₁₈ polyunsaturated fatty acids were minor components. These fatty acid distributions closely resemble those found in marine eustigmatophytes but are quite distinct from those found in most other algal classes. These microalgae also contain long-chain saturated and unsaturated monohydroxy fatty acids. Two distinct types of hydroxy fatty acids were found: a series of saturated α-hydroxy acids ranging from C₂₄ to C₃₀ with a shorter series of monounsaturated α-hydroxy acids ranging from C₂₆ to C₃₀ together with a series of saturated β-hydroxy acids ranging from C₂₆ to C₃₀. The latter have not previously been reported in either marine or freshwater microalgae, although C₃₀ to C₃₄ midchain (ω-18)-hydroxy fatty acids have been identified in hydrolyzed extracts from marine eustigmatophytes of the genus Nannochloropsis, and C₂₂ to C₂₆ saturated and monounsaturated α-hydroxy fatty acids have been found in three marine chlorophytes. These findings have provided a more complete picture of the lipid distributions within this little studied group of microalgae as well as a range of unusual compounds that might prove useful chemotaxonomic markers. The functions of the hydroxy fatty acids are not known, but a link to the formation of the lipid precursors of highly aliphatic biopolymers is suggested.     

RENEWAL OF GLYCEROL IN THE VISUAL CELLS AND PIGMENT EPITHELIUM OF THE FROG RETINA

The renewal of glycerol in the visual cells and pigment epithelium of the frog retina was studied by autoradiographic analysis of animals injected with [2-³H]glycerol. Assay of chloroform:methanol extracts showed that the labeled precursor was used mainly in lipid synthesis, although there was also some utilization in the formation of protein. Radioactive glycerol was initially concentrated in the myoid portion of rods and cones, indicating that this is the site of phospholipid synthesis in visual cells. The glycogen bodies (paraboloids) of accessory cones were also heavily labeled, suggesting the diversion of some glycerol into glycogenic pathways. In the pigment epithelium, only the oil droplets became significantly radioactive. The outer plexiform layer (which contains the visual cell synaptic bodies) and the cone oil droplets gradually accumulated considerable amounts of labeled material. Within 1–4 h, labeled molecules began to appear in the visual cell outer segments, evidently having been transported there from the myoid portion of the inner segment. Most of these were phospholipid molecules which became distributed throughout the outer segments, presumably replacing comparable constituents in existing membranes. In rods only, there was also an aggregation of labeled material at the base of the outer segment due to membrane biogenesis. These highly radioactive membranes, containing labeled molecules of lipid and protein, were subsequently displaced along the rod outer segments due to repeated membrane assembly at the base. The distribution of radioactivity supported the conclusion that membrane renewal by molecular replacement is more rapid for lipid than it is for protein.     

THE RELEASE OF BRAIN FREE FATTY ACIDS DURING ISCHAEMIA IN ESSENTIAL FATTY ACID-DEFICIENT RATS

—The release of free fatty acids and especially of free arachidonic acid occurring in rat brain during ischaemia has been studied in essential fatty acid-deficient animals. Free fatty acid levels are lower in essential fatty acid-deficient brains before and especially after 5 min of ischaemia. The percentage of arachidonic acid, in respect of total free fatty acids, is similar in both control and essential fatty acid-deficient brains before ischaemia (0 min), but is greatly reduced in deficient brains in respect of controls after ischaemia (5 min). Total levels of free arachidonic acid are thus greatly reduced after ischaemia in the brain of essential fatty acid-deficient rats. Focussed microwave irradiation of the brain, a technique which instantaneously kills the animals, and which was used for the determination of brain basal levels of free fatty acids and free arachidonic acid, does not per se modify brain lipid and fatty acid composition.     

樟科植物中脂肪酸成分的分布

本文报道了樟科11个属49种种子油和‘种果肉油的脂肪酸成分。其中樟族和木姜子族的各属种(滇新樟除外)种子油脂肪酸成分以 C_(10)、C_(12)和 C_(14)饱和或烯酸为主。而厚壳桂属、润楠和楠属种子油脂肪酸成分主要为棕榈酸、油酸和亚油酸。山苍子、天目木姜子、大果山胡椒、山橙、红脉钓樟、鸭公树和锈叶新木姜子种子油中的 C_(10)、C_(12)和 C_(14)烯酸经分离鉴定均是顺式Δ~4烯酸。     

KINETICS OF CELL RENEWAL, CELL MIGRATION AND CELL LOSS IN THE HAIRLESS MOUSE DORSAL EPIDERMIS

Forty hairless mice were given injections of tritiated thymidine every 4th hour during 10 days. At 24 hr intervals groups of four mice were killed. The numbers of labelled basal and differentiating cells were determined by autoradiography with a stripping film technique. To determine the background activity skin sections from uninjected control mice were subjected to the same stripping film procedure. Another group of hairless mice was given one single pulse labelling with tritiated thymidine. The number of labelled mitoses was scored for 12 hr after the injection. At 10, 12 and 15 hr after the injection, the numbers of labelled basal and differentiating cells were also determined. A mathematical model of cell population kinetics in the epidermis has been suggested. The results of different simulations on this model were compared with the observed results. The curve of mean grain counts under continuous labelling increased from day to day with two well-defined plateaux. The percentage of all labelled cells increased rapidly up to the 3rd day, and thereafter the curves gradually flattened off. When basal cells and differentiated cells were considered separately the labelling index of the basal cells increased rapidly for the first 3 days and then flattened off at the 100% level on the 5th day. The labelling index of the differentiating cells was low during the first 3–4 days. Then a steep increase in the percentage of labelled differentiating cells was seen, but the curve flattened off again close to the 100 % level after the 7th day. The labelled mitosis curve had its maximum 5 hr after the thymidine injection. The curve fell again to almost zero at 12 hr. Ten, 12 and 15 hr after the injection, 6, 7 and 7% respectively of the labelled cells were found in the spinous layer. It was concluded that three grains over each nucleus could be used as lower limit for considering a cell as labelled. On this basis, tritiated thymidine injections every 4th hour can be considered as continuous labelling.     

STIMULATION OF FUNDULUS BY HYDROCHLORIC AND FATTY ACIDS IN FRESH WATER,AND BY FATTY ACIDS,MINERAL ACIDS,AND THE SODIUM SALTS OF MINERAL ACIDS IN SEA WATER

1. Fundulus heteroclitus was found to be a reliable experimental animal for studies on chemical stimulation in either fresh or sea water. 2. The response of Fundulus to hydrochloric, acetic, propionic, butyric, valeric, and caproic acids was determined in fresh water, while the same acids plus sulfuric and nitric, as well as the sodium salts of the mineral acids, were tested in sea water. 3. Stimulation of Fundulus by hydrochloric acid in fresh water is correlated with the effective hydrogen ion concentration. Stimulation by the n-aliphatic acids in the same environment is correlated with two factors, the effective hydrogen ion concentration and the potential of the non-polar group in the molecule. However, as the number of CH₂ groups increases the stimulating effect increases by smaller and smaller amounts, approaching a maximum value. 4. Stimulation of Fundulus by hydrochloric, sulfuric, and nitric acids in sea water is correlated with the forces of primary valence which in turn are correlated with the change in hydrogen ion concentration of the sea water. The n-aliphatic acids increase in stimulating efficiency in sea water as the length of the carbon chain increases, but a limiting value is not reached as soon as in fresh water. 5. Only a slight difference in stimulation by hydrochloric acid is found in sea water and in fresh water. However, there is a significant difference in stimulation by the fatty acids in fresh and in sea water, which is partly explained by the different buffering capacities of the two media. It is to be noted that in the same environment two different fish, Fundulus and Eupomotis, give different results, while the same fish (Fundulus) in two different environments responds similarly to mineral acids but differently to fatty acids. These results illustrate that stimulation is a function of the interaction between environment and receptors, and that each is important in determining the response. 6. Stimulation by sodium chloride, nitrate, and sulfate is correlated with equivalent concentrations of the salts added to sea water, or with the forces of primary valence. Although the threshold for stimulation by the salts is considerably higher than for the acids, the efficiency of stimulation by the salts is greater.