THE ACTION OF EXCESS OF VITAMIN A ALCOHOL ON THE FINE STRUCTURE OF RAT DERMAL FIBROBLASTS

Rat dermal fibroblasts were grown as monolayers, and changes in the fine structure of the cells that occurred during 12 hr incubation in a medium containing protein and excess of retinol (vitamin A alcohol) were studied by electron microscopy. There is little change during the first 6 hr, although some of the nuclei have highly convoluted membranes. During the subsequent 3 hr, there is some disorganization of the mitochondrial cristae; the cisternae of the rough-surfaced endoplasmic reticulum diminish in number; and the amount of smooth membranous material and free ribosomes increases. There is a rapid decline in the respiratory activity of the cells after 6 hr exposure to the vitamin. It is concluded that the primary action of excess of retinol is to cause alterations in the membranes of the cells and that these alterations affect the functions of the mitochondria and endoplasmic reticulum.     

QUANTITATIVE STUDY OF NUCLEIC ACIDS AND PROTEINS IN THE SHOOT APEX OF SINAPIS ALBA DURING TRANSITION FROM THE VEGETATIVE TO THE REPRODUCTIVE CONDITION

Quantitative changes in DNA, histone, RNA, and total protein have been measured in meristematic cells during floral evocation.² A single 22-hr, long-day exposure induced two-month-old vegetative plants of Sinapis alba to flower. Periodic collections of shoot apices were made and stained with Schiff's reagent (DNA), azur B (RNA), alkaline fast green (histone), and naphthol yellow S (total protein). The two-wavelength method was used for DNA and histone measurements and the one-wavelength, two-area procedure was chosen for RNA and total protein determinations. The DNA and histone amounts per cell decreased to a minimum value 34 hr after treatment, and most of the nuclei shifted from 4C to 2C values. DNA and histone quantities paralleled each other from 34–46 hr, after which time the histone values continued to increase and the DNA values decreased. The RNA values increased rapidly after treatment as did the total protein quantities, after a slight decrease at 34 hr concurrent with the 4C to 2C cell population shift. The significance of these events is discussed in relation to the changes which were previously described in the shoot apex of Sinapis in transition to flowering.     

THE EARLY ACTION OF THE FLORAL STIMULUS ON MITOTIC ACTIVITY AND DNA SYNTHESIS IN THE APICAL MERISTEM OF XANTHIUM STRUMARIUM

Vegetative plants of Xanthium strumarium (a short-day species) were induced to flower by exposure to a single 16-hr long night. By cutting off the induced leaf (half-expanded leaf) at various times, it was established that, by 8 hr after the end of the long night, a sufficient amount of floral stimulus had reached the meristem to induce a flowering response. The following sequence of events occurred in both the peripheral and central zones of the apical meristem of induced plants: 1) a rise in the mitotic index beginning at 28 hr after the end of the long night and culminating at 36 and 56 hr; 2) a stimulation of DNA synthesis starting at 32–36 hr and reaching a maximum at 60 hr; 3) an increase in nucleolus diameter starting at 32 hr. The cell population in the meristems of both vegetative and induced plants displayed a similar distribution, with about 80 % of the nuclei with the 2C amount of DNA. The comparison of the kinetic data concerning the mitotic index and DNA synthesis indicated that one of the early effects of the floral stimulus in the peripheral and central zones was the release in mitosis of cells whose nuclei were in the postsynthetic (G₂) phase of the mitotic cycle. In the pith-rib meristem, the following events were recorded: 1) a stimulation of DNA synthesis starting at 20 hr; 2) a rise of the mitotic index beginning at 28 hr; 3) the vacuolation and elongation of cells starting at 48 hr. All these events occurred well before the initiation of bract and flower primordia, which began at 96 and 136 hr, respectively. Neither stimulation of mitotic activity nor flowering occurred in the meristems of plants subjected to a long night interrupted at its midpoint by a 5-min light break. The results are discussed in relation to the early events which are known to occur in the meristems of other photoperiodic species in transition to flowering.     

THE EFFECTS OF LIGHT ON THE DEVELOPMENT OF THE CELLULAR SLIME MOLD ACRASIS ROSEA

No fruiting of the NC-18 isolate of Acrasis rosea occurs in cultures maintained in continuous light or in continuous dark. The use of different food organisms does not alter the aforementioned behavior. The time at which fruiting occurs in this isolate can be regulated by administering stimulatory light followed by a dark period. Mature sorocarps are formed approximately 14 hr after the termination of light and the start of darkness. Within this 14-hr interval aggregation and sorocarp development occur. After about 6 hr of dark incubation, NC-18 amebae, previously stimulated by light, form a few weak aggregation centers. After 8 hr of dark incubation there are numerous aggregation areas, large in size and deep rose in color. By 10 hr the aggregations are quite compact and firm in appearance, and between 12 and 14 hr late aggregations, sorogens and, finally, mature sorocarps are formed. The minimum dark period, i.e., the minimum time that is required in darkness (for cultures previously stimulated with light) to obtain at least some fruiting within the 14-hr developmental period, is 7–8 hr for NC-18 and 5–6 hr for Tu-26. Maximum numbers of sorocarps form when cultures are given 10–11 hr of uninterrupted dark. Light-stimulated cultures of NC-18 placed in darkness and interrupted by a 10- or 30-min exposure to wide-spectrum blue or cool white fluorescent light an hour prior to the minimal dark period exhibit a 4–5 hr-delay in fruiting when returned to darkness and inspected at intervals following the second irradiation. Growth and fruiting of NC-18 occurred with purified food sources of each of five different species of Chlorella and with the alga Stichococcus bacillaris. This is apparently the first report of the utilization of algae as food sources by a cellular slime mold. Fruiting of NC-18 was readily arrested by lowering the relative humidity to 40–45%. This change in the moisture content of the surrounding air induced microcyst formation. Growth on buffered medium occurred in the entire pH range tested, 3.5–7.6, but fruiting occurred only between pH 5.0 and 6.6.     

THE ULTRASTRUCTURAL MORPHOLOGY AND ONTOGENY OF OAT COLEOPTILE PLASTIDS

Structurally similar proplastids occur in the shoot, scutellum, and root of the oat embryo at the start of germination. These proplastids follow several pathways of differentiation, depending on their location within an organ and on previous exposure to light. During the first 24 hr of germination morphologically similar amyloplasts are formed from the preexisting proplastids in most of the cells of the seedling. After about 24 hr in the light, unique chloroplasts begin to develop in a subepidermal ring of small cortical parenchyma cells in the coleoptile and give the organ a pale green color. At 48 and 72 hr the coleoptile chloroplasts and etioplasts are conspicuously different from the corresponding leaf plastids in morphology and ontogeny but contain typical photosynthetic grana and prolamellar bodies. Study of the ontogeny of plastids in the epidermal and nongreening parenchymal regions of dark grown coleoptiles shows that these plastids undergo significant losses in starch content, and some increase of membranes within the plastid, related to the age of the cell. Light has little effect on the structure of these plastids. It is suggested that the ontogeny of all the plastid types of the oat seedling begins with a common precursor—a relatively simple proplastid that is present at the time of germination. Starch grains showing two distinct types of erosion, apparently enzymatic, were observed in oat coleoptile plastids. In one type (grooved appearance) the starch grains are consistently associated with plastid membranes, while in the other type (irregular, spiny appearance) the starch grains are associated with the plastid stroma only. We suggest that there are two enzyme systems for metabolizing starch in oat plastids—one membrane-bound and the other free in the stroma.     

Function of coated membranes and multivesicular bodies during membrane regulation in stimulated exocrine pancreas cells

Summary Stimulation of secretion by pilocarpine results in a 70% loss of zymogen granules from pancreatic acinar cell during the first hr after injection of the drug. In previous work (Geuze and Poort, 1973), we found that the amount of membrane stored in the surface of the microvilli and of the numerous infoldings present in highly stimulated cells, increases during the first 2 hr and then decreases again during the 3rd hr after stimulation, concurrently with maximal endocytosis of sorbitol-[su14C].Further observations on the fine structure of stimulated cells at various time intervals after injection of pilocarpine showed that during the first hr numerous smooth vesicles and multivesicular bodies (mvb's) appear in the apical cytoplasm, while the number of coated vesicles and their relative total volume increase significantly 3 hr after stimulation.By infusion of ferritin in the pancreatic duct system in vivo and application of cytochemical techniques (osmium impregnation, electron microscope autoradiography and acid phosphatase cytochemistry) it could be established that after stimulated exocytotic secretion, redundant apical cell membrane is withdrawn by at least two routes: 1) During the initial rapid increase of the amount of apical cell membrane, withdrawal is accomplished by interiorization of luminal invaginations into smooth endocytotic vesicles, which in turn give rise to mvb's by infolding and subsequent fission of their limiting membrane. 2) Once the bulk of stored secretion granules has been discharged, endocytotic coated vesicles become gradually more prominent as carriers for redundant cell membrane. The contents of endocytotic structures ultimately become incorporated in residual bodies, suggesting lysosomal degradation of cell membrane prior to eventual reutilization.Coated vesicles also originate by pinching off from mature Golgi cisternae and condensing vacuoles. A possible function of the coated membranes in the concentration of exportable protein within forming secretory granules is discussed.     

CHANGES IN THE ACTIVITY OF ACID PHOSPHATASE AND THE APPEARANCE OF METAMORPHOSING POTENCIES OF THE LARVAE OF THE ASCIDIAN, HALOCYNTHIA RORETZI

In the swimming phase of the larvae of the ascidian, Halocynthia roretzi , changes in the activity of acid phosphatase (AcP-ase) were studied cytochemically with respect to the appearance of metamorphosing potencies. The AcP-ase activity in the larvae before and soon after hatching is weakly visible only in and around the nuclei of the epithelial, muscular and notochordal cells. 6 hr after hatching the enzyme activity begins t o appear weakly in the microblasts around the proximal end of the notochordal sheath, whereas the activities which were found in the previous stages disappear. In the larvae which passed for 12 hr after hatching, the activity of AcP-ase is distinctly shown in the microblasts and also in the other 2 mesodermal cells, meso- and macro- blasts. The microblasts of this stage are closely attached to the notochordal sheath at the proximal end. At the same time, many large granules which appear similar to lysosomes are found in the microblast by an electron microscopy. The 6th hour's larvae after hatching can be induced slowly to resorb its tail by the treatment with a nile blue solution, but the time which it takes for tail resorption is gradually shortened depending on the age of the larva up until 12 hr after hatching.
From these results, i t was concluded that the appearance of the AcP-ase activity in the microblasts was parallel with the appearance of the potency of metamorphosis of the larvae after hatching. Possible roles of the microblasts at onset of meta- morphosis would seem to play a role in the rupture of the notochordal sheath and in the succeeding regression of the tail tissues.     

A plaque assay for titration of alfalfa looper nuclear polyhedrosis virus in a cabbage looper (TN-368) cell line

This is the first report of plaque formation by a pathogenic insect virus. Trichoplusia ni (TN-368) cells overlaid with medium containing 0.6% methyl cellulose continued to multiply, developed into monolayers, and produced plaques after infection with alfalfa looper nuclear polyhedrosis virus. Viral polyhedral inclusion bodies were first observed 24 hr after exposure of cells to virus, and plaques continued to increase in size for 72 hr. Two different types of plaques were observed: one in which all cells had many polyhedra in their nuclei, and another in which few cells had inclusion bodies. When virus from either plaque was injected into T. ni larvae, they died of typical nuclear polyhedrosis virus disease. The assay was reproducible, and plaque numbers were related to virus concentration.     

THE ULTRASTRUCTURE AND HISTOCHEMISTRY OF A NEMATODE-INDUCED GIANT CELL

The development of giant cells induced by the nematode Meloidogyne in tomato roots has been followed under controlled growth conditions and the ultrastructure and histochemistry of these structures have been examined. Entry of the nematode larvae into the roots took place within 24 hours; giant cell formation started on the 4th day and involved breakdown of the cell walls accompanied by thickening of a surrounding giant cell wall and an increase in density and area of the cytoplasm. The nuclei increased in number by simultaneous mitosis throughout a single giant cell. The peak of cytoplasmic density was reached after moulting and during egg production. The rate of protein synthesis in the giant cell is correlated with the rate of growth of the nematode. The giant cell wall is a thick, irregularly surfaced structure which contains all the normal polysaccharide components of a cell wall. The cytoplasm is rich in protein and RNA and contains mitochondria, proplastids, Golgi bodies, and a dense endoplasmic reticulum. The nuclei are large and irregular in shape and contain large nucleoli and a number of Feulgen-positive bodies scattered irregularly along the nuclear envelope. The nucleolus contains RNA and fat as well as Feulgen-positive granules which are revealed after treatment with ribonuclease. It consists of a dense outer cortex surrounding a much lighter central core and is connected at times with the Feulgen-positive bodies in the nucleus. Speculation is provided on the role of these bodies in cytoplasmic protein synthesis.     

OBSERVATIONS ON THE FINE STRUCTURE OF THE MACRONUCLEUS OF TOKOPHRYA INFUSIONUM

The macronucleus in Tokophrya infusionum is composed of numerous Feulgen-positive chromatin bodies (about 0.5 µ in diameter) which appear in thin sections as a dense spongework, homogeneous throughout. The same appearance characterizes metaphase chromosomes of higher forms. Some chromatin bodies of the macronucleus were found to possess a highly organized structure in certain old organisms. This structure appears in cross-sections as a honeycomb and in longitudinal sections as parallel lines about 120 A in diameter evenly spaced (about 230 A). As far as is known this is the first time a regular structure has been found in bodies of chromosomal character at the dimensional level presently explored by electron microscopy. The demonstration that OsO₄ can preserve order in chromatin material is another significant aspect of these findings.     

THE DISORGANIZATION OF HEPATIC CELL NUCLEOLI INDUCED BY ETHIONINE AND ITS REVERSAL BY ADENINE

The structure of nuclei and nucleoli of hepatic cells after short-term ethionine administration was investigated with the electron microscope. By 1½ hr after the injection, a distinct alteration occurred in the nucleoli which was characterized by the appearance of electron-opaque masses in the nucleolonema. After 6–8 hr, the nucleoli showed partial fragmentation into small, dense masses. Large aggregates of interchromatinic granules appeared in the nucleoplasm. Condensation of chromatin became prominent in the nucleoplasm particularly along the nuclear membrane. By 12 hr almost complete fragmentation of nucleoli had occurred. The administration of adenine or methionine at 4 hr prevented the development of nucleolar changes. Also, adenine administration at 8 hr after ethionine completely reversed the nucleolar lesion by 12 hr. After methionine administration at 8 hr, many nucleoli showed incomplete reconstruction with many twisted ropelike structures when viewed 4 hr later. Identical structures were found when adenine was given at 8 hr, and animals were sacrificed 2 hr later. On the basis of this observation, the simplified structures of nucleoli found 2 hr after adenine or 4 hr after methionine appeared to be precursors of the nucleolonema. It is suggested that nucleoli show at least two basic reaction patterns to inhibitors of RNA synthesis, one typified by actinomycin D and one by ethionine.     

ULTRASTRUCTURAL CHANGES IN CELLS OF PEA EMBRYO RADICLES DURING GERMINATION

Radicles (1 mm long) of pea embryo axes, excised from dormant seeds and from seeds soaked for 80 min, 8, 12, and 48 hr, were fixed in glutaraldehyde and postfixed in OsO₄. The nucleus was only slightly changed by 48 hr soaking, but cytoplasmic organelles underwent developmental changes. There was proliferation of endoplasmic reticulum, the appearance of dictyosomes, and an inward migration of lipid bodies. Most of these changes were observed within 8 hr after soaking started. Plastids, mitochondria, protein bodies, and protein crystalline bodies were also identified and their developmental changes were followed.     

Changes in wall and internal structure of allomyces-resistant sporangia during germination

The electron microscope was used to examine changes which take place in wall, as well as in internal, structure during germination of mature resistant sporangia of Allomyces neo-moniliformis. When the resistant sporangia are first placed in water to initiate germination, nuclei, mitochondria, and endoplasmic reticulum are not evident, though after the sporangia have been in water for more than 30 min all of these structures become visible. At this time no cracks are evident in the resistant sporangial wall and the cell membrane appears highly convoluted. Within the next 30 min the outer wall splits and the inner wall expands considerably as the protoplast increases in volume. At the same time the cell membrane straightens out, apparently in response to the protoplasmic expansion. The “cementing substances” begin to dissolve about this time so that 1 1/2 hr after placement in water the outer wall is completely separated from the inner wall which now acts as the cell wall. Cleavage appears to be initiated by the invagination of the cell membrane and by the appearance of segments of endoplasmic reticulum with filled vesicles at one end. Between 2 1/2 and 3 hr after placement in water zoospores are released.     

AN AUTORADIOGRAPHIC STUDY OF THE 3H-URIDINE AND 3H-THYMIDINE INCORPORATION IN THE REGENERATING MOUSE LIVER

An autoradiographic study was made of the ³H-uridine incorporation into RNA and DNA in nucleus and cytoplasm of parenchymal cells in the regenerating liver of the mouse after a pulse time of 2 hr. After a decreased uptake of precursor into the parenchymal nucleus during the first 6 hr compared with the normal value, incorporation increased and was maximal at 36 hr; normal values were restored at 72 hr. The cytoplasmic labelling, after an initial small decrease, reached a maximum at 12 hr; this changed to normal 48 hr after hepatectomy. RNase-digestion of the liver sections left a small incorporation in both nucleus and cytoplasm: presumably DNA. This incorporation is maximal at 12 hr over the nucleus and at 24 hr over the cytoplasm. After a 2 hr pulse of ³H-thymidine, there was a marked uptake of the precursor into DNA about 24 hr after hepatectomy. This was maximal at 48 hr and reached normal values at 72 hr. A small amount of incorporation of ³H-thymidine into DNA was seen immediately after the operation, and this population of weakly labelled nuclei was still rather large 72 hr later.     

AXOPLASMIC TRANSPORT IN THE OPTIC NERVE AND TRACT OF THE RABBIT

The axonal transport of labelled proteins was studied in the optic system of adult rabbits after an intraocular injection of [³H]Ieucine. It was demonstrated that the precursor was incorporated into protein, which was transported along the axons of the retinal ganglion cells. Intraocularly injected puromycin inhibited protein synthesis in the retina and markedly inhibited the appearance of labelled protein in the optic nerve and tract. It was further demonstrated by intracisternal injection of [³H]leucine that an intraocular injection of puromycin did not affect the local protein synthesis in the optic nerve and tract. Cell fractionation studies of the optic nerve and tract showed that the rapidly migrating component, previously described as moving at an average rate of 110-150 mm/day, was largely associated with the microsomal fraction. About 40 per cent of the total protein-bound radioactivity in this component was found in the microsomal fraction and about 15 per cent was recovered in the soluble protein fraction. Most of the labelled material moving at a rate of 1-5-2 mm/day was soluble protein. The specific radioactivity of this component was about ten times greater than that of the fast one. In the slow component about 50 per cent of the radioactivity was found in the soluble protein fraction and about 10 per cent of the radioactivity was recovered in the microsomal fraction. Radioautography demonstrated incorporated label in the neuropil structures in the lateral geniculate body as early as 4-8 hr after intraocular injection. The labelling of the neuropil increased markedly during the first week, and could be observed after 3 weeks.     

THE SHOOT APICAL ONTOGENY OF THE PICEA ABIES SEEDLING. I. ANATOMY,APICAL DOME DIAMETER,AND PLASTOCHRON DURATION

Developing embryos in immature Picea abies seeds already have well-delineated shoot apical meristems with clearly evident cytohistological zonation. During early seedling development the zonation characteristic of gymnospermous apical meristems is attained. Seedling development is also accompanied by an approximately threefold increase in apical dome diameter. The latter approaches a steady state about 140 days after germination. Seedlings display a spiral phyllotaxis consisting of a contact parastichy system, usually of the primary Fibonacci series. As the seedlings age and apical domes enlarge, higher Fibonacci number-pairs characterize their phyllotaxis. Mathematical analysis of the relation between cumulative leaf number and age revealed that the length of the plastochronic time interval declines from about 18.5 hr to 5.7 hr as seedling age increases from 20 to 140 days.     

THE ULTRASTRUCTURE AND PHYSIOLOGY OF PROTEIN BODIES AND LIPIDS FROM HYDRATED DORMANT AND NONDORMANT EMBRYOS OF SETARIA LUTESCENS (GRAMINEAE)

Embryo protein bodies in S. lutescens have a uniform matrix and contain no globoid or crystalloid inclusions. Their digestion pattern consists of peripheral pitting, and the occurrence of vesicles along the margin of the formed invaginations suggests a controlled release mechanism. Protein bodies from both dormant and nondormant embryos were observed to be in all stages of digestion. Total protein decreases in both during the first 6 hr of hydration. Lipids do not decrease in hydrated dormant florets although a significant reduction does occur in germinating nondormant florets. Dormancy, therefore, may involve a mechanism that does not control processes already derived (protein body digestion) but does inhibit processes first begun by germination.     

THE EFFECT OF ESTRADIOL ON THE CELL KINETICS IN THE UTERINE AND CERVICAL EPITHELIUM OF NEONATAL MICE

The effect of estradiol-17β on the length of the various phases of the cell cycle was studied in the neonatal mouse uterine, and cervical epithelium. A double labelling method was used, and in addition labelled mitoses were counted. In the uterus proper, estradiol shortens the length of the total cell cycle, T_c, from 17-9 hr to 15-7 hr, and the duration of S phase, T_s, from 6–7 to 5-1 hr 6 hr after estradiol treatment. 12 hr after estradiol treatment, T_c is shortened to 7-4 hr and T_s to 4–5 hr. The shortening of T_c at 12 hr is mainly due to an effect on T_G1, which is shortened from 8–55 hr in untreated animals to 1–8 hr in estradiol treated animals. The T_c of cervix epithelium cells in untreated animals was found to be 21-8 hr. After treating the mice for 6 hr with estradiol the T_c was now increased to 47 hr and further to 61 -2 hr following 12 hr treatment with the hormone. T_s increases from 8-3 hr to 15-2 hr following 6 hr estradiol treatment, and to 15-4 hr after 12 hr treatment. The effect is most pronounced in T_G1, which is lengthened from 10–95 hr in untreated animals to 28-1 hr and 43 hr, respectively, in animals treated for either 6 or 12 hr with estradiol.     

THE USE OF TRITIATED THYMIDINE IN THE STUDY OF TISSUE ACTIVATION DURING GERMINATION IN ZEA MAYS

Stein, O. L. (Montana State U., Missoula), and H. Quastler. The use of tritiated thymidinein the study of tissue activation during germination in Zea mays. Amer. Jour. Bot. 50(10): 1006-1011. Illus. 1963.—Corn embryos were exposed to H³thymidine at various times during the first 80 hr of germination. An analysis of labeled nuclei was made from autoradiographs, and the number and position of mitoses were recorded. Those tissues which approach maturity during embryogeny (root cap, coleorhiza, scutellur node) are first to resume DNA synthesis (30 hr after soaking). No mitoses were observed in these tissues. In shoot and root, mitoses usually precede DNA synthesis, indicating that the nuclei of the dormant embryo have a DNA value of 4C(twice the diploid DNA) or more. The shoot begins its activity much later than the root (50 hr). The shoot apex was the last region to boeomo active, some 70 hr after initiation of the soaking treatment.     

PROTEIN UPTAKE INTO MULTIVESICULAR BODIES AND STORAGE GRANULES IN THE FAT BODY OF AN INSECT

The fat body in Calpodes ethlius (Lepidoptera, Hesperiidae) takes up protein from the blood throughout the larval stage before pupation. Depending upon the phase of development, the protein appears in multivesicular bodies, in large storage granules, and in structures of intermediate form. There are three phases in the 8 days of the last larval stage; the first devoted to growth (molting to 66 hr), the second to synthesis for storage or export (M + 66 to M + 156 hr), and the third to preparation for pupation (M + 156 to pupation at M + 192 hr). From M + O to M + 156 and from M + 180 to M + 188 hr, protein is taken up into multivesicular bodies. Larger MVB's form a continuous series with the protein granules formed from M + 162 to M + 180 hr. Blood proteins increase in concentration and amount from M + 66 to M + 156 hr at the same time as the fat body cells have a high rate of incorporation of amino acids and a structure appropriate for protein synthesis. During granule formation, both amino acid incorporation and blood protein concentration decrease. Since foreign proteins injected into the blood appear in the granules, they are probably made mainly from sequestered blood. Protein uptake involves two stages: concentration between the cells, and ingestion in pinocytotic vesicles. The vesicles fuse to become MVB's or storage granules, depending upon their rates of growth and the addition of lytic enzymes. Since MVB's do not accumulate in the fat body and since many of them contain acid phosphatase and appear empty, they are presumed to be concerned in protein turnover.