POPULATION DENSITY EFFECTS ON GROWTH IN CULTURE OF THE EDIBLE SNAIL HELIX ASPERSA VAR. MAXIMA

The effects of population density on the growth of H. aspersaMÜller var. maxima under controlled environmental conditionswere examined. Inhibitory effects on snail growth and maturityresulting from increased population density, between 100–800snails m^–2 of floor area, were observed for a range ofcontainer cleaning frequencies. At all population densities,enhanced snail growth was observed when the frequency of containercleaning was increased to a two-day interval. No significantdifferences were recorded, following 19 weeks growth, betweenfinal mean weights of snails from containers cleaned less frequently.The lowest snail mortality was consistently recorded at thelowest population density in the most frequently cleaned containers.At all snail population densities three phases of growth wereobserved: (a) lag (0–5 weeks), (b) rapid (6–15 weeks)and (c) stable (16 weeks and over). During the first three weeksof growth, high population density had a positive effect ondiet consumption, food conversion efficiency and snail growth.Adverse population density effects increased progressively duringphase (b), typically following 9 weeks growth. Juvenile snailstransferred from high to low population densities during phase(b) continued to exhibit slower growth rates associated withhigh population densities. Food conversion efficiency of snailsin all treatments decreased throughout the experimental periodbut with no overall effect of container cleaning frequency apparent.Inherent growth variability of sibling snails was unaffectedby population density or container cleaning frequency. The importanceof the results for intensive snail culture is discussed. (Received 23 June 1994; accepted 1 December 1994)     

Pyrimidine Nucleotide Biosynthesis in Vinca rosea Cells: Changes in the Activity of the de novo and Salvage Pathways during Growth in a Suspension Culture

Marked changes in the activity of the ‘de novo’and ‘salvage’ pathways of pyrimidine biosynthesisduring growth of Vinca rosea cells in a batch suspension culturewere observed. The activity of these pathways was investigated by determiningthe contribution of ¹⁴C of [2-¹⁴Cluracil, 12-¹⁴Cluridine. and[6-¹⁴Clorotate to the cell constituents and by measuring theactivity of the several enzymes of these pathways. During the lag phase of the culture, ‘uracil-’ and‘uridine-salvage’ pathways made the predominantcontribution to nucleotide biosynthesis, but, following theinitiation of cell division, the ‘de novo’ pathwayfor nucleotide biosynthesis operated appreciably. These results suggest that nucleotide synthesis during cellgrowth in a suspension culture can be divided into two stages:a ‘turnover stage’, during the lag phase of cellgrowth, and a ‘true biosynthetic stage’, which isinitiated in the cell division phase.     

Distribution of membrane-bound and free ribosomes in growing yeast.

1. The distribution of membrane-bound and free ribosomes was investigated in stationary as well as in growing yeast cells. the relative amount of free ribosomes varies with the growth phase of the cell culture. During the duplication phases of the cell, relative maxima of free ribosomes can be found. However, the absolute amount of free ribosomes is fairly constant during the growth of the cells. 2. Membrane-bound ribosomes show lower polypeptide synthesis activity in a cell-free, poly (U)-dependent system than free ribosomes. 3. There is no difference in the distribution pattern of free and membrane-bound ribosomes in growing yeast cells of different ploidy. 4. A turnover between free and membrane-bound ribosomes is suggested to be in agreement with the hypothesis of Branes and Pogo ((1975) Eur. J. Biochem. 54, 317-328).     

Control of macromolecular synthesis in proliferating and resting Syrian hamster cells in monolayer culture. I. Ribosome function

The rate of protein synthesis per cell in cultured hamster embryo fibroblasts in the stationary growth phase falls to about one third of the rate in the exponential growth phase. This reduction can be entirely accounted for by the following observations: (1) the average cell in stationary phase contains about one-half the number of ribosomes per cell compared to the average cell in exponential phase; (2) only two thirds of the ribosomes are bound to polysomes in stationary phase, while nearly all of the ribosomes are polysome-bound in exponential phase. In stationary phase, ribosomes which are polysome-bound function with the same efficiency and produce proteins of approximately the same average length as in exponential phase. Experimental findings are presented which suggest that the generation of a higher proportion of free ribosomes in stationary phase in not due to a limitation in messenger RNA, but to a decreased attachment probability of ribosomes to messenger RNA.     

Changes in the Endoplasmic Reticulum During Sieve Element Differentiation in Triticum aestivum

The changes in structure of the endoplasmic reticulum (ER) andits associations with other cell components have been studiedin differentiating protophloem sieve elements of root tips ofTriticum aestivum. In the young sieve elements single ER cisternaebearing ribosomes are dispersed in the cytoplasm. As differentiationprogresses ER increases in amount while a small proportion ofit aggregates into stacks or becomes associated with the nuclearenvelope and the mitochondria. These modifications occur inthe last two sieve elements containing ribosomes and coincidewith most dramatic changes in the degenerating nucleus. Stacksconsist of relatively few ER cisternae and may be encounteredfree in the cytoplasm or applied to the nuclear envelope. Electron-densematerial accumulates between the contiguous cisternae of thestacks. ER-attached ribosomes persist even in nearly maturesieve elements, but their pattern of arrangement becomes changed.The structural evidence indicates that only a few highly degradedER elements are retained in fully mature sieve elements. Triticum aestivum, root protophloem, sieve elements, endoplasmic reticulum, differentiation     

Modulation of the number of membrane-bound auxin-binding sites during the growth of batch-cultured tobacco cells

We studied the modulation of the number of membrane-bound naphthaleneacetic acid (NAA)-binding sites during the growth cycle of tobacco cells in batch culture. Both cell number and specific NAA-binding increased exponentially, but at different rates and for different periods. This caused a characteristic modulation of the number of binding sites per cell during the growth cycle: During the first day of the lag phase this number decreased; in the exponential phase it rose markedly, and in the stationary phase it was constant.Abbreviations MES 4-morpholinoethanesulphonic acid - NAA 1-naphthaleneacetic acid     

Studies on the Growth in Culture of Plant Cells: I. GROWTH PATTERNS IN BATCH PROPAGATED SUSPENSION CULTURES

Techniques are described for following increases in total cellnumber, fresh weight and dry weight, and changes in mean cellsize, and in the relative number of free cells to cell aggregatesduring the growth of batch-propagated suspension cultures oftissues derived from several species of angiosperms. When totalcell number is plotted against time it is seen that there canbe distinguished in sequence a lag phase, phases of acceleration,maximum rate, and negative acceleration of cell division and,finaly, a stationary phase. Studies with Parthenocissus tricuspidatacrown-gall tissue, growing in a synthetic liquid medium, haveshown that the total cell production per culture in the firstinstance is limited by nitrate supply rather than by the supplyof other inorganic ions, sucrose supply aeration, or the releaseof endogenous inhibibors. Studies, particularly with Acer pseudoplatanustissue, have shown that during the period of high cell-divisionrate, mean cell size reached its minimum value and average numberof cells per cell aggregate its maximum value. Cell separationdoes not occur to a significant extent until cell-division activityhas almost ceased and it is dependent upon cell expansion. Thebalance between cell division and cell expansion determinesthe ‘cellular unit’ composition of the cultures.Refinement of the control of growth patterns in plant suspensioncultures calls for further study of the ‘conditioning’of media, of factors which limit the duration of the periodof high mitotic activity, and of the conditions necessary forfull and rapid cell expansion.     

NUCLEOLAR AGING IN TETRAHYMENA DURING THE CULTURAL GROWTH CYCLE

Nucelolar morphology was studied by electron microscopy in control and actinomycin D-treated populations of Tetrahymena pyriformis (W) during the cultural growth cycle. Nucleoli exhibit an "aging" cycle concomitant with the cultural growth cycle, but independent of the individual cell cycle. Four different stages in the course of this aging process have been defined. Stage 1 occurs upon inoculation (low number of cells per milliliter) and lasts through lag and accelerating growth phases. In this stage, many small nucleoli are found at the nuclear periphery. In stages 2 and 3, nucleolar fusion begins. Stage 2 dominates the first half of logarithmic growth, and stage 3 dominates the second half. In late decelerating growth phase, the nucleoli enter stage 4. In this stage, only a few large nucleoli are present and these are apparently inactive in ribosome production. In stationary phase, where total RNA remains constant, only stage 4 nucleoli are present. The relative preponderance of granular vs. fibrous components in the nucleoli changes during this cycle, the granular component dominating stage 1 nucleoli and the fibrillar, stage 4 nucleoli. There is a shortening of the intermediate nucleolar stages in the treated cultures; fusion occurs early and is now pronounced. Not enough ribosomes accumulate to carry the treated cultures through the number of generations equivalent to those of the control, which produces a premature stationary phase.     

Studies on the Growth in Culture of Plant Cells: VIII. THE PRODUCTION OF ETHYLENE BY SUSPENSION CULTURES OF ACER PSEUDOPLATANUS, L.

Sycamore cell suspension cultures in a synthetic medium releaseethylene; during a 24-day incubation period a single culture(initial volume 70 ml) produces c. 4 µ moles. There isa very sharp peak of ethylene production between day 10 andday 14 of culture; at the peak of production c. 2 nmoles ethyleneare released per million cells in 24 h. Evidence is presentedthat 2,4-D enhances ethylene production independently of itseffects on culture growth. Under the standard conditions of culture (250-ml Erlenmeyerflasks closed with aluminium foil and containing 70 ml cellsuspension) the concentration of ethylene in the gas phase ofthe cultures rises above 10 ppm. No evidence was obtained thatthis ethylene is inhibitory to culture growth or that a criticallevel of ethylene is necessary to initiate cell division incultures at a critically low cell density. The low rate of ethylene release by stationary phase culturesis temporarily enhanced by the addition of various solutes andfurther depressed by dilution with water.     

Protein synthesis during the transition from the resting to the growing state in suspension cultures of Paul's Scarlet rose cells

Cells derived from Paul's Scarlet rose ( Rosa sp. ) were grown in the chemically defined medium of Nesius. When a stationary phase culture was diluted with fresh medium, growth was initiated after a pronounced lag period. DNA replication, as revealed by thymidine labeling and autoradiography, did not begin until 36 h, and mitotic figures were not observed until 48 h after dilution. A 10–15 fold increase in the rate of protein synthesis occurred during the lag period. This was brought about by a 3.5 fold increase in the amount of ribosomal RNA per cell, plus a doubling of both the percentage of ribosomes that are present as polyribosomes and the average number of ribosomes per polyribosome. The spectrum of polypeptides synthesized by these cells during the lag and early log periods of growth was examined. Polyribosomes were extracted from the cells at intervals preceding and accompanying the initiation of proliferative growth. The polyribosomes were translated in a wheat germ cell-free protein synthesizing system and the ³⁵S-methionine-labeled translation products were separated on polyacrylamide slab gels and by 2-dimensional gel electrophoresis. Comparatively few differences were observed between stationary phase, lag phase and log phase cells in terms of the spectrum of polypeptides synthesized in vitro. However, these various phases of the growth cycle could be characterized by a relatively high rate of synthesis of a few specific polypeptides. That is, while most proteins are synthesized throughout the growth cycle and even in non-growing cells at approximately the same relative rates, there are a few variable proteins whose synthesis marks a particular phase of the growth cycle.     

Studies on the Growth in Culture of Plant Cells: XIX. CHANGES IN THE LEVELS OF FREE AND MEMBRANE-BOUND POLYSOMES DURING THE GROWTH OF ACER PSEUDO PLA TAN US CELLS IN BATCH SUSPENSION CULTURE

Techniques have been established which give reproducible yieldsof total and of free and bound polysoines from cultured sycamorecells liarvested at intervals throughout the cycle of growthfollowed in batch culture. High levels of polysoines build upin the cells during lag phase and persist into early exponentialgrowth. Later in the growth cycle, although the ribosomal materialper unit volume of culture continues to rise, the content percell of ribosomes and polysomes progressively declines untilthe cells enter the stationary phase. There is also a characteristicpattern of change in the relative proportions of free and boundpolysomes throughout the growth cycle of the cultures. Bothfractions contain different but significant levels of ribosomalsubunits and monomers. The RNAs released from the free polysoineshad a greater amount of poly(A) sequences than that from thebound polysomes. These findings are discussed in relation tothe changing metabolic activities of the cells which occur duringtheir progress through batch culture.     

Regulation of RNA synthesis in plant cell culture: delayed synthesis of ribosomal RNA during transition from the stationary phase to active growth

Ribosomal RNA synthesis was studied during the early phases of growth activation in a cell suspension culture derived from peanut (Arachis hypogaea, L.) cotyledon. Upon dilution from stationary phase, these cells show a characteristic lag of 3 days before the commencement of cell division. An analysis of the nature of RNA synthesized during this early period of growth showed that the cells obtained immediately upon dilution from stationary phase synthesize primarily messenger RNA and essentially no ribosomal RNA. The synthesis of ribosomal RNA is delayed for about 24 hr after which it rises sharply resulting in a 2- to 3-fold accumulation of ribosomal RNA per cell during the subsequent 24-hr period. Both the messenger RNA and the ribosomal RNA were characterized by their cellular localization; by sucrose and CsCl gradient analyses, and by the determination of their base ratios.It would appear that a major facet of the lag phase in the cell growth is the diversion of a significant part of the RNA biosynthetic apparatus from the synthesis of messenger RNA to that of ribosomal RNA.     

The Relationship between Growth Rate, Differentiation and Alkaloid Accumulation in Cell Cultures

The relationship between alkaloid content, growth rate and differentiationwas investigated in seven solanaceous species in culture. Evidencewas obtained for an inverse correlation between the growth phaseon the one hand and alkaloid content and cell organization (aggregation)on the other. Linear phase and stationary phase cells of Daturainnoxia Mill, incorporated DL-(1_–14C)ornithine-HCl differentiallyinto protein and alkaloids with proportionately more label foundin alkaloids in the stationary phase cells. On transferringcallus of D. innoxia from high auxin medium (containing 10–6M 2, 4–D) to low auxin medium (containing 10–6 M,2, 4–D), the alkaloid content dropped concomitant witha decrease in chlorophyll content and an increase in growthrate and friability. The nature of the interrelationships betweengrowth, differentiation and accumulation are considered. Key words: Tropane alkaloids, Accumulation, Growth, Differentiation     

Ammonium Triggers Uptake of NO-3 by Chenopodium rubrum Suspension Culture Cells and Remobilization of their Vacuolar Nitrate Pool

Photoautotrophic cell suspension cultures of Chenopodium rubrumrequire high concentrations of nitrate and ammonium. Duringthe growth phase total NH₄⁺ and the greater portion of NH₃^–were consumed. During the stationary phase nitrate uptake continuedbut at a substantially smaller rate than during the growth phase.During growth the bulk of the absorbed N was incorporated intoprotein, the amount of which was then maintained constant untilsenescence. NH₃^– was accumulated upon transition betweenthe growth and the stationary phase. NH₃^–, like the freeamino acids, was deposited in the vacuole but, unlike thesecompounds, could not be remobilized upon transfer of the cellsinto N-free medium. Readdition of NH₄⁺ to the medium, however,resulted in a mobilization of the vacuolar NH₃^–-pool.Reutilization of both vacuolar N-storage pools must have beenaccomplished by recycling from the vacuole to the cytoplasmbecause N-metabolizing enzymes could not be detected in isolatedvacuoles. Transfer of the cells of the stationary phase intomedium containing NH₃^– and NH₄⁺ resulted in an inductionof nitrate uptake by the cells, but only after a lag phase of4–5 days. It is conceivable that NH₄⁺ induces NH₃^–-translocatingsystems in the plasmalemma and in the tonoplast. (Received December 19, 1988; Accepted March 2, 1989)     

THE FATE OF MITOCHONDRIA DURING AGING IN TETRAHYMENA PYRIFORMIS

During the growth cycle of Tetrahymena pyriformis the mitochondria undergo changes in position, number, and structure. Ciliates in the logarithmic growth phase possess elongated mitochondria which are aligned along the plasma membrane and are closely associated with the kinetosomes and kinetodesmata. Mitochondria appear to divide across the long axis at this time, resulting in two or more products. Throughout this phase of growth mitochondrial divisions keep pace with cytokinesis so that the population of mitochondria remains at essentially the minimal level. As the ciliates enter the stationary growth phase the mitochondria increase in number, become oval to spherical in shape, and some migrate into the cytoplasm. Intramitochondrial masses of various configurations appear at this time. Some of the mitochondria lying in the cytoplasm become incorporated into vacuoles. Within these vacuoles either a single mitochondrion appears or several mitochondria may be seen along with other cytoplasmic structures. Later in the stationary growth phase the contained mitochondria are dense and the tubules are more compact than normal. Various stages in disorganization of the mitochondria are observed in a single large vacuole. Cytochemical tests reveal the presence of acid phosphatase, suggesting that hydrolysis of the vacuolar contents occurs. Lipid droplets increase in number during the middle and late stationary phase of growth. These events are interpreted as being associated with the normal process of aging in T. pyriformis.     

Ultrastructure of Gum-resin Ducts in Cashew (Anacardium occidentale)

In Anacardium occidentale L., the gum-resin ducts in the primaryphloem of the stem develop schizogenously. Appearance of anintercellular space amongst the densely stained cells signalsthe initiation of a duct. During this process, the middle lamella,appears in places, as an electron-opaque area. The epitheialcells bordering the duct are oval and have convex inner tangentialwalls which are without any plasmodesmata, although they areabundant on radial and outer tangential walls. The cytoplasmof the epitheial cells is rich in rough endoplasmic reticulum(ER), free ribosomes, polysomes, mitochondria with swollen cristae,plastids with occasional osmiophilic inclusions, dictyosomesand vesicles. Osmiophilic material has been observed in thedilation of the cisternae of dictyosomes and also in vesicles.Sometimes, the osmiophilic material aggregates and forms largemembrane-bound globules. The globules fuse with the plasmalemmaat the inner tangential wall, and presumably the contents aredeposited in the space between the protoplast and the wall.This material passes through the loose matrix of the wall intothe duct. Some of the epitheial cells of the mature duct show‘dark’ cytoplasm, degraded organelles and occasionallyhigh vacuolation; ultimately they are lysed and their remainscollect in the duct. Anacardium occidentale L., cashew, gum-resin ducts, epithlial cell, dictyosome, osmiophilic material, ultrastructure     

CHANGES IN CELL FINE STRUCTURE DURING LENS REGENERATION IN XENOPUS LAEVIS

Changes at the level of cell fine structure have been studied during lens regeneration in the toad, Xenopus laevis, where cornea gives rise to the new lens. The transformation of these cells may be divided into three phases. (1) In the cornea, flattened cells become cuboidal and rough endoplasmic reticulum increases in amount. (2) In the new lens vesicle, cisternae of the rough ER break down into vesicles, smooth-walled vesicles and free ribosomes increase in number, and mitochondria can become enlarged and irregular, then centrally attenuated. Rudimentary cilia form. (3) As new lens fibers form, ribosomes become very numerous and low density fibrous elements and dense clumps appear in the cytoplasm. These phases are accompanied by marked nucleolar changes. The changes during the 3rd phase are similar to changes in the lens during normal development. The first two phases show an unexpected morphological complexity.     

SIEVE-ELEMENT ONTOGENY IN THE ROOT OF EQUISETUM HYEMALE

Roots of Equisetum hyemale L. var. affine (Engelm.) A. A. Eat. were fixed in glutaraldehyde, postfixed in osmium tetroxide, and sieve elements of various ages were examined with the electron microscope. Young sieve elements are distinguished by their position within the vascular cylinder and by the presence of numerous refractive spherules, which originate within dilated portions of the endoplasmic reticulum (ER). Early in development, the sieve-element walls undergo a substantial increase in thickness. This is followed by the appearance of massive ER aggregates in the cytoplasm and then by a phase involving stacking and sequestering of the remaining ER. Nuclear degeneration is initiated shortly after the appearance of the ER aggregates. The chromatin condenses into masses of variable size along the inner surface of the nuclear envelope. The envelope then ruptures and chromatin is released into the cytoplasm. During the period of nuclear degeneration, mitochondria and plastids undergo structural modification, while components such as dictyosomes, microtubules, and ribosomes degenerate and disappear. The remaining cytoplasmic components assume a parietal position in the cell, leaving the lumen of the cell clear in appearance. At maturity, the plasmalemma-lined sieve element contains plastids, mitochondria, some ER, and refractive spherules. At this time many of the refractive spherules are discharged into the region of the wall. Pores between sieve elements occur largely on the end walls. During pore development, tubules of ER apparently traverse the pores, but because of the presence of massive callose deposits in the material examined, the true condition of mature pores could not be determined. The connections between mature sieve elements and pericycle cells are characterized by the presence of massive wall thickenings on the pericycle-cell side. Plasmodesmata in the wall thickening are matched by pores on the sieve-element side. Ontogenetic and cytoplasmic factors argue against use of the term “companion cell” for the vascular parenchyma cells associated with the sieve elements.     

Sieve-Element Ontogeny in the Aerial Shoot of Equisetum hyemale L.

The aerial shoots of Equisetum hyemale L. var. affine (Engelm.)A. A. Eat. were examined with the electron microscope as partof a continuing study of sieveelement development in the lowervascular plants. Young E. hyemale sieve elements are distinguishablefrom all other cell types within the vascular system by thepresence of refractive spherules, proteinaceous bodies whichdevelop within dilated portions of the endoplasmic reticulum(ER). Details of cell wall thickening differ between protophloemand metaphloem sieve elements. Following cell wall thickeningthe ER increases in quantity and aggregates into stacks. Shortlythereafter, nuclear degeneration is initiated. During the periodof nuclear degeneration some cytoplasmic components-dictyosomes,microtubules and ribosomes-degenerate and disappear, while organellessuch as mitochondria and plastids persist. The latter undergostructural modifications and become parietal in distribution.Eventually the massive quantities of ER are reduced, leavingthe lumen of the cell clear in appearance. At maturity the plasmalemma-linedsieve element contains a parietal network of tubular ER, aswell as mitochondria, plastids, and refractive sphemh At thistime many of the spherules are discharged into the region ofthe wall. Sieveelement pores occur in both lateral and end walls.At maturity many pores are traversed by large numbers of ERmembranes. The metaphloem sieve elements of the mid-internodalregions apparently are sieve-tube members. The connections betweenmature protophloem sieve elements and pericycle cells are associatedwith massive wall thickenings on the pericyclecell side.     

Ribosome degradation in growing bacteria

Ribosomes are large ribozymes that synthesize all cellular proteins. As protein synthesis is rate-limiting for bacterial growth and ribosomes can comprise a large portion of the cellular mass, elucidation of ribosomal turnover is important to the understanding of cellular physiology. Although ribosomes are widely believed to be stable in growing cells, this has never been rigorously tested, owing to the lack of a suitable experimental system in commonly used bacterial model organisms. Here, we develop an experimental system to directly measure ribosomal stability in Escherichia coli. We show that (i) ribosomes are stable when cells are grown at a constant rate in the exponential phase; (ii) more than half of the ribosomes made during exponential growth are degraded during slowing of culture growth preceding the entry into stationary phase; and (iii) ribosomes are stable for many hours in the stationary phase. Ribosome degradation occurs in growing cultures that contain almost no dead cells and coincides with a reduction of comparable magnitude in the cellular RNA concentration.