Cell Synchrony Techniques. II. Analysis of Cell Progression Data

Abstract CHO cells which have been sorted by mitotic detachment, centrifugal elutriation and fluorescence activated cell sorting have been followed for up to 14 hr by flow cytometry to examine their progression characteristics. Mathematical modelling techniques were used to provide quantitative estimates of the cell-cycle parameters. Mitotic detachment gives an 11.2-hr cycle time with mean transit times T_G1, T_s and T_G2M equal to 3.2, 5.6 and 2.4 respectively. Cells prepared by central elutriation in an early G₁ state have a 14-hr cycle time with T_G1, T_s and T_G2M of 5.7, 6.0 and 2.3 hr. Populations prepared by centrifugal elutriation enriched in early S and late S and G₂M have transit times of 2.7, 5.9 and 1.6 hr and 4.9, 6.7 and 2.1 hr with cycle times of 11.2 and 13.2 hr respectively. Cell sorting for a G₁ population gives transit times of 9.8, 8.0 and 3.6 for an overall 21.4-hr cycle time.     

EFFECTS OF PHOTOCYCLES AND PERIODIC AMMONIUM SUPPLY ON THREE MARINE PHYTOPLANKTON SPECIES. II. AMMONIUM UPTAKE AND ASSIMILATION1

The relative influence of the photoperiod and of periodic ammonium pulses in entraining the cell division cycle in nitrogen-limited cyclostat cultures differs dramatically in Hymenomonas carterae Braarud and Fagerl, Amphidinium carteri Hulburt and Thalassiosira weissflogii Grun. We examined how each species processes an NH₄⁺ pulse at various times during the cell cycle and the L/D cycle. Rates of NH₄⁺ uptake and changes in cellular concentrations of NH₄⁺, free amino acids, and protein were examined after the addition of an NH₄⁺ pulse. Depletion of NH₄⁺ from the medium occurred earlier when the pulse was given at the beginning of the light period than at the beginning of the dark period in H. carterae and A. carteri. Depletion took longer in the T. weissflogii cultures and the kinetics were similar during both stages of the photocycle in this species. Similarly, the temporal phasing and maximum pool sizes varied with timing of the NH₄⁺ pulse in H. carterae and A. carteri but complete assimilation was relatively rapid. More persistent pools of NH₄⁺ and free amino acids accumulated in T. weissflogii, and the patterns of assimilation varied little as a function of the timing of the pulse with respect to the photocycle. Although nitrogen metabolism occurred rapidly in nitrogen-limited H. carterae and A. carteri, the entrainment of the cell division cycle by the photoperiod resulted in a large degree of uncoupling between completion of nitrogen assimilation and cell division. It is hypothesized that the strong entrainment of the cell division cycle of T. weissflogii by NH₄⁺ pulses results from a relatively slow rate of nitrogen metabolism.     

Centaurea forojuliensis,della sect. Jacea DC. s. str., nuova entità dal Friuli

Abstract

Centaurea forojuliensis, sect. Jacea DC. s. str., new entity from Friuli.—The new endemic entity Centaurea forojuliensis present in the marshes of Friuli (Northeastern Italy), is described. It is probably an ecotypical differentiation inside the polymorphic cycle of Centaurea Jacea due to the particular habitat. A taxonomic account of the whole cycle in the Southeastern Alps and Northadriatic carstic regions is also given.     

VARIATION IN MATING CALL ACROSS THE HYBRID ZONE BETWEEN THE FIRE-BELLIED TOADS BOMBINA BOMBINA AND B. VARIEGATA

Three components of mating call (pulse duration, cycle length, and fundamental frequency) were measured and six diagnostic enzyme loci scored across the hybrid zone between the toads Bombina bombina and B. variegata. All three call components differ significantly, but only cycle length is diagnostic. The clines in call coincide with those for enzymes, and have similar widths. This suggests that there is no strong selection on any of these characters. There are significant correlations between electrophoretic markers and call components, but these are no stronger than would be expected if the electrophoretic loci and the genes causing mating call were neutral. The selection differential on the call is no greater than 6% of the difference in mean cycle length between the two taxa. There is a substantial increase in the variance of cycle length in the center of the zone, suggesting that a small number of loci are involved (≈ three). Recombination between these loci will hinder the evolution of reinforcement and may partly be responsible for the lack of premating isolation between B. bombina and B. variegata.     

Regulation of biosynthesis of secondary metabolites. I. Biosynthesis of chlortetracycline and tricarboxylic acid cycle activity

In the course of submerged cultivation of low-production and industrial production strains of Streptomyces aureofaciens, the activity of enzymes of the tricurboxylic acid cycle was studied. The activities of citrate synthase (EC 4.1.3.7), aconitate hydratase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), fumarate hydratase (EC 4.2.1.2), and malate dehydrogenase (EC 1.1.1.37) were estimated spectrophotometrically in cell-free preparations. In the growth phase, mainly the initial reactions of the cycle were active with both strains. In production-phase, the activities of enzymes in the low-production strain were 2–5 × higher than in the production strain. Benzylthioeyanate, at a concentration of 5 × l0^?5M, stimulated chlortetracycline production of both strains with accompanying decrease in activity of the enzymes of the tricarboxylic acid cycle. The role of the tricarboxylic acid cycle in control of chlortetracycline biosynthesis is discussed.     

Description and life cycle of Leuctra iliberis sp. n. from Southern Spain (Plecoptera,Leuctridae)

The nymphs and adults of a new species of the genus Leuctra Stephens, 1835, L. iliberis are described and figured.

The life cycle is presented as result of monthly sampling during an annual cycle in the Sierra Nevada mountains (southern Spain).     

Variations in biochemical parameters of Heterocapsa sp. and Olisthodiscus luteus grown in 12:12 light:dark cycles I. Cell cycle and nucleic acid composition

The division cycle of two phytoplankton species, Olisthodiscus luteus and Heterocapsa sp. was studied in relation to a 12:12 light:dark cycle. Batch cultures in exponential phase were sampled every three hours during 48 hours. Cell number, cellular volume and DNA and RNA concentrations were measured. Microscopic observations of the nuclei of Heterocapsa sp. were also performed. In both species, cell division took place in the dark. In Heterocapsa sp., DNA and RNA showed a similar diel variability pattern, with synthesis starting at the end of the light period, previously to mitosis and cytokinesis. In O. luteus. Major RNA synthesis occurred during darkness, and DNA was produced almost continuously. Both species presented different values and diel rhythmicity on the RNA/DNA ratios.     

DIATOM MINERALIZATION OF SILICIC ACID. II. REGULATION OF Si (OH)4 TRANSPORT RATES DURING THE CELL CYCLE OF NAVICULA PELLICULOSA1

Synchronized populations of Navicula pelliculosa (Bréb.) Hilse show a 10-fold increase in Si(OH)₄ transport rate during traverse through the cell division cycle. The transport activity pattern is similar to a “peak enzyme.” Kinetic analysis showed there was a significant change in K_s values, indicating increased “affinity” for Si(OH)₄ as cells neared maximal uptake rates. However, the dramatic changes in transport rate at various cell cycle stages were also reflected by alterations in the V_max, values of the transport process, suggesting a change in the number of functional transport “sites” in the plasma membrane. Cells in the wall forming stage, arrested from further development by Si(OH)₄ deprivation, maintained high transport rates for as long as 7 h. The rates decreased rapidly if protein synthesis were blocked or if Si(OH)₄ was added, the latter allowing the cells to traverse the rest of the cycle. The half-life of the transport activity ranged from 1.0 to 2.2 h when protein synthesis was inhibited at various cell cycle stages and during the natural decline of activity late in the cycle. The transport system appears to be metabolically unstable as is typical for a “peak protein.” The rise in transport rate through the cell cycle did not depend on the presence of Si(OH)₄ in the medium; therefore, the transport system does not appear to be induced by its substrate. The rise in transport is also observed in L:D synchronized cells developing in the presence of Si(OH)₄; neither does the transport system appear to be derepressed. The transport rate was strongly cell cycle-stage dependent; the data appeared to fit the “dependent pathway” model proposed by Hart-well to explain oscillations in enzyme synthesis during the cell cycle.     

Waterlogging duration: Interspecific comparison of Leptospermum scoparium (Forst et Forst.f.), Acacia melanoxylon (R. Br.), Nothofagus cunninghamii (Hook.) and Eucalyptus obliqua (L’Herit)

Abstract The effect of the duration of waterlogging on the pre‐dawn water potential, gas exchange, biomass accumulation and survival was investigated on four species, Leptospermum scoparium (Forst et Forst.f.), Acacia melanoxylon (R. Br.), Eucalyptus obliqua (L’Herit) and Nothofagus cunninghamii (Hook.). These species co‐occur, but are restricted to particular microsites. The three waterlogging treatments applied to potted seedlings were: four cycles of 15 days, two cycles of 30 days or one 60‐day cycle, followed by an equal period in freely drained conditions. Water potential, gas exchange, biomass accumulation and survival were not significantly affected by the duration of waterlogging in L. scoparium or A. melanoxylon. With increased waterlogging duration, N. cunninghamii had progressively decreased survival, had less biomass accumulation in all waterlogging treatments and more negative water potential after the 60‐day waterlogging cycle. In contrast, E. obliqua had low survival under the 15‐day waterlogging cycle treatment, although survival, biomass accumulation and maximum net photosynthesis were decreased by all waterlogging cycle treatments. Water potential was significantly lower (more negative) in E. obliqua than other species after the 30‐ and 60‐day waterlogging cycle treatments only. When exposed to oxygen deficit in hypoxic nutrient culture for 5 days, L. scoparium had 100% survival and maximum net photosynthesis was not affected. Acacia melanoxylon had 80% survival and decreased photosynthesis from 2 days of exposure onwards. Nothofagus cunninghamii and E. obliqua had 70% and 30% survival, respectively, and their photosynthesis was significantly depressed after 1 day of exposure to hypoxic conditions. Relative tolerance of the species examined to waterlogging and hypoxia was consistent with adaptation to conditions of seasonal variation in water table height and soil oxygenation concentration observed at the microsites occupied by the respective species. The results indicate that duration of waterlogging may be a major determinant in the microsite distribution of the co‐occurring species investigated.     

Microbiology and chemistry of acid lakes in Florida: II. Seasonal relationships

The microstratification of the metalimnetic community in Lake Cisó was followed through the diel cycle by means of a fine sampler with syringes spaced at 3 cm intervals. Populations were sharply stratified. The uppermost part of the metalimnion was occupied by the rotifer Anuraeopsis fissa. Next, layers of the ciliate Coleps hirtus and the flagellate Cryptomonas phaseolus were found. Finally, purple sulfur bacteria appeared at the bottom part of the metalimnion. Although in lower abundances, characteristic populations of ciliates were found at each depth: Strombidium inhabited zones with oxygen, while Plagiopyla and Metopus lived at depths with sulfide. Several members of the community moved vertically about 20 cm during the diel cycle. Cryptomonas performed larger vertical migrations of 40 cm. This organism concentrated at the upper metalimnion during the day, and dispersed through the lower metalimnion during the night. At night, Cryptomonas lived in an environment without oxygen and with sulfide concentrations up to 0.6 mM.     

A New Species of Psychrophilic Basidiomycetous Yeasts Leucosporidium fasciculatum sp. nov.

A psychrophilic yeast with a basidiomycetous developmental cycle and properties corresponding to the genusLeucosporidium Fell et al. was isolated from the fruiting body of the edible spring mushroom Gyromitra esculenta Pers. picked near Moscow. However, the isolate differed from all Leucosporidium species described to date in a number of characteristics. The results of the study of the developmental cycle and of the cultural, morphological, physiological, and biochemical properties of the new isolate, strain KBP Y-3696, allow it to be assigned to a new species of the genus Leucosporidium.     

Lotharella amoeboformis sp. nov.: A new species of chlorarachniophytes from Japan

The ultrastructure, morphology and life cycle of a new chlorarachniophyte alga collected from Okinawa in Japan have been studied. The life cycle of this alga consists of amoeboid, wall‐less round, coccoid and flagellated cells in culture condition; however, the coccoid and flagellate cells are very rare. The pyrenoid ultra‐structure of this alga is the same as that of a previously described species, Lotharella globosa. Since pyrenoid ultrastructure has been adopted as the main criterion for the generic classification of the chlorarachniophytes, the present alga is placed in Lotharella. However, the present alga has a dominant amoeboid cell stage and a reduced walled‐cell stage in the life cycle, while in L. globosa, the walled‐cell stage is dominant and there is no amoeboid cell stage. Therefore the present alga is described as a new species of Lotharella: Lotharella amoeboformis Ishida et Y. Hara sp. nov.     

Mutants with continuous microcycle conidiation in the filamentous fungus Fusarium solani f. sp. pisi

Summary A method of repeated mutation induction and subsequent selection was used to obtain mutants with microcycle conidiation, mc, in Fusarium solani f. sp. pisi. Wild-type, wt, Fusarium has a filamentous (hyphal) growth in a three-stage, conidium—hypha—conidiophore (phialid) vegetative life cycle. The first major mutation induced (by means of the mutagen MNNG) changed the wild-type strain 1–3^- from producing both macro- and microconidia to the mutant strain 19-P2^-, producing only microconidia but preserving the three-stage life cycle. When strain 19-P2^- was exposed to a similar mutagenic treatment mutants without the hyphal stage were obtained. In these mutants the microconidia germinate to form phialids directly, which in turn produce new conidia, thus accomplishing the life cycle as a two-stage, conidium-conidiophore, microcycle. The mc mutants were stabilized after reisolation through back-crossing to the wt of complementary mating type. Mc growth of the mutants is continuous under stable conditions, proceeding uninterrupted to the stationary stage in liquid as well as on solid medium. Mc colonies on solid medium are small and soft, well suited to replication by means of velvet or paper. Large numbers of mature, synchronized conidia are easily obtained from stationary-stage submerged cultures. Sequential mutations are of interest for studies of morphogenetic evolution. The ahyphal mutants make this group of eukaryotes well suited for genetic engineering and biotechnology.     

Trypanosoma cruzi: Biological Characterization of Clones Derived from Chronic Chagasic Patients. II. Quantitative Analysis of the Intracellular Cycle1

The complete intracellular cycle of five cloned stocks of Trypanosoma cruzi was quantified. Marked but stable interclonal differences were found in the length of the pre-replicative lag period (18.2-34.2 h), amastigote doubling lime (8.6-21.5 h), and duration of the complete intracellular cycle (96-215 h). Strong correlations were demonstrated between these characteristics as well as to the growth rate of the epimastigote stage of the same clones grown in liquid medium. These data demonstrate that the marked heterogeneity of the natural population of T. cruzi extends to the intracellular cycle of the parasite and has important implications for our understanding of Chagas’ disease.     

Biology and the B.Ed. Degree

The dispersion of animals is one aspect of ecology that lends itself to laboratory simulation. Photographs of the Tenebrionid beetle Catamerus rugosus were taken at different stages in its life cycle. Students were asked to relate these to real life and carry out a statistical analysis to determine the degree of dispersion of the animals. The photographs are shown and the results demonstrate a change in dispersion throughout the life cycle.     

GROWTH AND NITROGEN FIXATION OF THE DIAZOTROPHIC FILAMENTOUS NONHETEROCYSTOUS CYANOBACTERIUM TRICHODESMIUM SP. IMS 101 IN DEFINED MEDIA: EVIDENCE FOR A CIRCADIAN RHYTHM1

Trichodesmium sp. IMS 101, originally isolated from coastal western Atlantic waters by Prufert-Bebout and colleagues and maintained in seawater-based media, was successfully cultivated in two artificial media. Its characteristics of growth, nitrogen fixation, and regulation of nitrogen fixation were compared to those of natural populations and Trichodesmium sp. NIBB 1067. Results indicate that the culture grown in artificial media had nitrogen fixation characteristics similar to those when the culture is grown in seawater-based medium and to those of Trichodesmium sp. in the natural habitat. The study provides practical artificial media to facilitate the physiological studies of these important diazotrophic cyanobacteria, as well as the cultivation of other Trichodesmium species in future studies. Manipulations of the light/dark cycle were performed to determine whether or not the daily cycle of nitrogen fixation is a circadian rhythm. Cultures grown under continuous light maintained the cycle for up to 6 days. We demonstrated that the daily cycle of nitrogen fixation in Trichodesmium sp. IMS 101 was at least partially under the control of a circardian rhythm.     

The effect of lead on the cell cycle in the root meristem ofAllium cepa L.

Summary Allium cepa (L.) adventitious roots were treated with lead (2.5 mg of Pb²⁺ [from Pb(NO₃)₂] per dm³) for 30–72 h. The cell cycle was studied by pulse labeling with [³H]thymidine. Mitotic activity kinetics, occurrence of disturbed mitoses (c-mitoses), and level of DNA synthesis were examined. It was found that lead prolonged the cell cycle and that cells in two phases of the cycle, G₂ and S, differed in their sensitivity to lead. Cells in G₂ were more sensitive; lead lengthened their cycle by 216% and disturbed the course of cell division by causing c-mitoses. Cells in S phase were less sensitive. Their cell cycle was longer by 55%. They went through their G₂ phase without major disturbances, mitosis in these cells was normal. During treatment ofA. cepa with lead, its destructive effects on cells were exerted only during the first few hours (around 6 h) of incubation. That is when the inhibition of mitotic activity, numerous disturbances of cell division, a decline in the number of cells synthesizing DNA, and a lower level of DNA synthesis were observed. As the incubation continued, the above processes were found to return to normal. In the discussion, data are presented supporting the hypothesis that during the initial period of exposure ofA. cepa to lead, this metal enters both the root apoplast and symplast, exerting a destructive effect on cells, while later, lead penetrates only into the root apoplast, and in this way remains harmless to cells.     

Life Cycles of Leucocytozoon dubreuili Mathis and Leger, 1911 and L. fringillinarum Woodcock, 1910 (Haemosporidia: Leucocytozoidae)*

SYNOPSIS The development of Leucocytozoon dubreuili and L. fringillinarum was studied on successive days in simuliid and avian hosts. Sporogony of both parasites is completed in at least 5 species of sylvatic Simuliidae in a minimum of 4-5 days at 21 C. The pattern of development of the 2 species is similar but the size of the oocysts and the number of sporozoites differ. Sporozoites of L. dubreuili and L. fringillinarum were injected into uninfected robins (Turdus m. migratorius) and grackles (Quiscalus quiscula versicolor), respectively. Hepatic biopsies were performed on some of the injected birds. These and others were killed at intervals following inoculation and their tissues examined to detect stages of schizogony. Blood and macerated tissues from birds injected with sporozoites were transferred to uninfected birds to determine whether asexual stages would develop in the latter as a result of the inoculations. The 1st asexual cycle of L. dubreuili is completed in hepatic parenchymal cells in a minimum of 84 hr. Merozoites produced by the hepatic schizonts apparently follow one of 3 courses: invade hepatic parenchymal cells to initiate another cycle; penetrate blood cells and become gametocytes; penetrate tubular cells of the kidneys and grow into renal schizonts. The minimum prepatent period in infections with L. fringillinarum is 76 hr. The 1st asexual cycle occurs in hepatic parenchymal cells and in tubular cells of the kidney. A schizogonic cycle is completed in a minimum of 72 hr in the former and 96 hr in the kidney. Merozoites from the primary hepatic schizonts apparently give rise to (a) gametocytes; (b) secondary hepatic schizonts; (c) renal schizonts. Thus the schizogonic cycles of L. dubreuili and L. fringillinarum differ from each other and from those of L. simondi in ducks.     

The ultrastructure of Mychonastes ruminatus gen. et sp. nov., a new member of the Chlorophyceae isolated from brackish water

A new monotypic genus of the Chlorophyceae isolated from brackish water of the Chesapeake Bay, Maryland, U.S.A. is described as Mychonastes ruminatus. The alga is compared with similar members of the Oocystaceae. Based on the presence of a thick ruminate cell wall, the lack of discernible pyrenoids, and other vegetative characters, Mychonastes can be delineated from other green coccoid unicells.

The cellular life cycle of Mychonastes was studied by electron microscopy and compared to published studies of Chlorella. The genera are differentiated by wall and chloroplast structure. Mychonastes cell wall is sculptured and changes during the life cycle from a highly irregular appearance when young, to a less irregular state when mature. Mychonastes chloroplast lacks pyrenoids at all times. Chlorella has a smooth wall and pyrenoids which are apparent just after release from the mother cell until division. Organelles of both genera migrate in a similar pattern throughout the life cycle, with Mychonastes producing two or four autospores, and Chlorella producing four or more (rarely two).     

THE COMPARATIVE CELL CYCLE AND METABOLIC EFFECTS OF CHEMICAL TREATMENTS ON ROOT TIP MERISTEMS. I. IOXYNIL

In this study we investigated the cell cycle response of Vicia faba and Pisum sativum root tip meristems to ioxynil treatments at two concentrations, (10^−-4m and 10^−-6m ). After 24 h of treatment at 10^−-4m concentration, O₂ uptake and ATP concentrations were significantly reduced. The mitotic index was reduced and the cell cycle population position was shifted to indicate that previously inhibited cells reformed their nuclei and became tetraploid. Prolonged treatment at this concentration resulted in cell death. Treatment with ioxynil at 10^−-6m reduced the rate of entry into mitosis. Abnormal mitotic figures in all stages were observed, and the ploidy level of mitotically inhibited cells was doubled. These observations indicated that at 10^−-6m concentration ioxynil acts as a preprophase inhibitor, that is, it does not act directly on the mitotic apparatus but does affect processes on which mitosis depends.