Timing and Regulation of Nuclear and Cortical Events in the Cell Cycle of Tetrahymena pyriformis*

SYNOPSIS. Using continuous flow cultures based on the chemostat principle, we varied the cell generation times of the ciliate Tetrahymena pyriformis strain GL, from 4.9 to 22.2 hr and studied various parameters of the cell cycle at 28 C. These included: the duration of the periods required for oral morphogenesis, macronuclear division, cell division, G₁ S, and G₂. The size of individual cells was also measured. Independent of the growth rate, the period of oral morphogenesis occurred during the last 90 min of the cell cycle. In all cases macronuclear and cell divisions took place during the last part of these 90 min, and the final macronuclear separation occurred just before final cell separation. The S-period increased slightly, while the G₁ and G₂ both increased in roughly the same relative proportion to the increasing generation times. Slowly growing cells (generation time 20.5 hr) were shorter but broader and somewhat larger in volume than quickly growing cells (generation time 4.9 hr).     

Metabolism of lipid and carbohydrate in sea urchin spermatozoa

When the dry sperm of the sea urchin, Hemicentrotus pulcherrimus, were diluted 100 times in artificial sea water at 0°C and at 20°C, they became motile and the levels of ATP and creatine phosphate decreased rapidly. The level of ADP hardly changed, and the AMP level increased after the dilution. After the dilution, the respiratory rate at 2°C was almost one fifth that of 20°C. Both phospholipid and glycogen were used for the energy sources in sea urchin sperm. The level of phospholipid was 10-fold higher than that of glycogen in the dry sperm. The phospholipid level decreased after dilution at 20°C, though the level hardly changed at 0°C, suggesting that phospholipid was hardly metabolized the lower temperature. The level of α -glycerophosphate increased at 20°C after the dilution but did not change at 0°C. The level of glycogen decreased after the dilution, regardless of the temperature. The glycolysis was also activated after the dilution. Of the intermediates of the tricarboxylic acid cycle, the citrate concentration increased at 0°C and the malate concentration also increased at 0°C and especially strongly at 20°C.     

High-density photoautotrophic algal cultures: design, construction, and operation of a novel photobioreactor system

A photobioreactor system has been designed, constructed and implemented to achieve high photosynthetic rates in high-density photoautotrophic algal cell suspensions. This unit is designed for efficient oxygen and biomass production rates, and it also can be used for the production of secreted products. A fiber-optic based optical transmission system that is coupled to an internal light distribution system illuminates the culture volume uniformly, at light intensities of 1.7 mW/cm(2) over a specific surface area of 3.2 cm(2)/cm(3). Uniform light distribution is achieved throughout the reactor without interfering with the flow pattern required to keep the cells in suspension. An on-line ultrafiltration unit exchanges spent with fresh medium, and its use results in very high cell densities, up to 10(9) cells/mL [3% (w/v)] for eukaryotic green alga chlorella vulgaris. DNA histograms obtained form flow cytometric analysis reveal that on-line ultrafiltration influences the growth pattern. Prior to ultrafiltration the cells seem to have at a particular point in the cell cycle where they contain multiple chromosomal equivalents. Following ultrafiltration, these cells divide, and the new cells are committed to division so that cell growth resumes. The Prototype photobioreactor system was operated both in batch and in continuous mode for over 2 months. The measured oxygen production rate of 4-6 mmol/L culture h under continuous operation is consistent with the predicted performance of the unit for the provided light intensity.     

Chloroplast phosphoribulokinase associates with yeast phosphoriboisomerase in the presence of substrate

Pea chloroplastic phosphoribulokinase and yeast phosphoriboisomerase partition independently of one another in a two-phase polyethyleneglycol, dextran system, but apparent interaction is seen when ribose-5-phosphate is added to the two-phase system. It appears that the pea leaf of kinase recognizes yeast isomerase when it is carrying metabolite.     

Notes on the morphology,ecology and life cycles of Fukaurahydra anthoformis and Hataia parva (Hydrozoa,Athecata)

Fukaurahydra anthoformis and Hataia parva are solitary athecate hydroids occurring in northern Japan. New information on the external morphology, nematocysts, ecology, and life cycles of these species is presented. It is noteworthy that H. parva bears stenoteles, which are generally not found among the families of Filifera. Neither species produces free medusae. The eggs are fertilized in the female gonophores, from which unciliated larvae are released. These larvae do not swim and soon attach to a substrate. After attachment the larvae become covered by a sheath to form cysts. The cysts rest on a substrate without any outer change for several months. As the water temperature drops in autumn to early winter the cysts begin to hatch, forming tiny polyps after the larva creeps out from the chitinous sheath. Cyst formation proves to be common also in other solitary hydroids, most of which are inhabitants of cool or cold waters.     

Cell Cycling of Astrocytes and Their Precursors in Primary Cultures: A Mevalonate Requirement Identified in Late G1, but Before the G1/S Transition, Involves Polypeptides

The relationship between mevalonate and cell cycling was investigated in developing glial cells. Primary cultures of newborn rat brains were serum-depleted (0.1%, vol/vol) for 48 h on days 4-6 in vitro, then returned to 10% calf serum (time 0). After 48 h, 70-80% of the cells were glial fibrillary acidic protein (GFAP)-negative by indirect immunofluorescence; 79 +/- 7% were GFAP-positive after an additional 3 days. Serum shift-up resulted in 12 h of quiescence, and then by 20 h (S phase) in increased proportions of cells synthesizing DNA (from 15 +/- 6% to 75 +/- 4% by bromodeoxyuridine immunofluorescence at 12 h and 20 h, respectively) and rates of DNA synthesis (42 +/- 6 versus 380 +/- 32 cpm/micrograms of protein/h of [3H]thymidine uptake). Additional mevalonate (25 mM) for 30 min at 10 h reversed the inhibition of DNA synthesis apparent with mevinolin (150 microM), an inhibitor of mevalonate synthesis, present from time 0. Cycloheximide added simultaneously with mevalonate prevented this reversal of inhibition. To cause arrest at G1/S, cultures were exposed to hydroxyurea between 10 and 22 h. By 3 h after hydroxyurea removal, bromodeoxyuridine-labeled nuclei increased from 0% to 75 +/- 9%, and DNA synthesis increased 10-fold. Mevinolin failed to inhibit these increases. Thus, primary astroglial precursors stimulated to progress through the cell cycle express a mevalonate requirement in late G1, but before the G1/S transition. The effect of mevalonate was characterized further as being brief (30 min) and as requiring polypeptides.     

Commitment to the First Cortical Reorganization During Conjugation in Stylonychia mytilus: An Argument for Homology with Cortical Development During Binary Fission

The asexual nature of the first cortical reorganization of conjugation in Stylonychia was analyzed by comparing the effect of amputation performed at different stages of early conjugation to that performed on vegetative cells at different stages of the cell cycle. Amputation of vegetative cells delineated a point of commitment to binary fission at 0.51–0.57 of the cell cycle. Cells amputated before this point were induced to undergo the regenerative mode of asexual development, but those amputated after this point continued with binary fission. In parallel, during conjugation a similar commitment was made around the time of formation of tight mating-pairs: early conjugants amputated around this time might undergo regeneration, and those operated on after this stage continued with the first cortical reorganization as in typical conjugants. The two mates of a pair might differ in their response to amputation, suggesting that the timing of commitment to the first cortical reorganization is not related to the events of conjugation, but rather is individually determined in the vegetative cycle of the cells before they pair up in mating. These observations provide support for the notion that the first cortical reorganization of conjugants is homologous to the asexual mode of cortical development in dividers, according to the theory of developmental heterochrony in the sexual reproduction of hypotrichs. The timing of commitment to the first cortical reorganization was found to temporally correlate with the entrance of the micronuclei into meiosis. Since the first cortical reorganization can proceed without the micronucleus, this raises the possibility that initiation of micronuclear meiosis is closely coupled with, and may be determined by, the commitment to the first cortical reorganization.     

Life Cycle of Paramecium bursaria Syngen 1 in Nature

ABSTRACT. Studies were completed on the natural population density of Paramecium bursaria syngen 1 and on the life cycle stages to which the individuals belonged. Green paramecia were collected from two streams once every 20 days for over one year: 413 individuals on 26 collection dates in Mikumarikyo stream and 83 individuals on 23 collection dates in Momijidani-gawa stream. Individuals in nature did not maintain at a steady density but fluctuated greatly depending on the month. It seems that conjugation occurred from April to June in the Mikumarikyo stream and from May to June in the Momijidani-gawa stream. The appearance of individuals with mating ability might be related closely to increasing population so that sexual reproduction probably occurred near the peak of the population density. The 413 individuals from Mikumarikyo stream were examined to determine their position within the life cycle; 309 (74%) were immature, 55 (13%) were adolescent, and 49 (12%) were mature. No senile individuals were observed. The fraction of individuals with mating ability was generally less than 30% at any collection. Four mating types were observed occurring with about equal frequencies in mature individuals. The results show the frequencies of the recessive genes for mating types (a and b) are higher than for dominant genes (A and B). Of 83 individuals from Momijidani-gawa stream, 44 (52%) were immature, 21 (25%) were adolescent, and 18 (21%) were mature. Again, no senile individuals were observed. Because only two mating types were found, II and III (genotypes aaB- and aabb), it seems possible that the dominant gene A was rare or absent in the Momijidani-gawa population.     

Possible role of cell cycle-dependent morphology,geometry, and mechanical properties in tumor cell metastasis

Studies that examine the shear- and abrasion-sensitivity of proliferating cells are important in order to understand the behavior of hybridoma cells in bioreactor culture and metastasizing cancer cells in the bloodstream. Little is known about the link between morphology, structure, and mechanical properties of a given cell line, especially with respect to variations throughout the cell cycle. In our experiments with GAP A3 hybridoma cells, distinct cell morphologies were identified and correlated with phases of the cell cycle by video microscopic observation of synchronized cells, and of individual cells that were followed throughout their cell cycle. Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (apparent cell viscosity) properties of single cells. As the cell cycle progressed at 37°C, an increase in cell volume from 1400 μm³ to 5700 μm³ was accompanied by an increase in apparent cell viscosity from 430 poise to 12,000 poise, consistent with an accumulation of more cytoplasmic material in the “older” cells. Hybridomas are representative of the various leukemias derived from hemopoietic cells, and even though as a whole, they appeared to be rather shear-insensitive, the wide range of property values demonstrates that a given cell line cannot be characterized by a single value for any one property, and that properties must be related to the cell cycle when considering proliferating cells. It is interesting to see if distinct stages in the metastatic sequence of events might correlate with any of these physical features of the cell cycle, irrespective of cell type or cell line. For example, the cytokinetic doublet could represent a fragile structure that may fail and produce cell death under fluid-shear conditions that would not affect the cells at any other stage in the cell cycle. Identifying such cell cycle-dependent features in metastasizing cancer cells could lead to a better understanding of the metastatic process and to possible clinical treatments directed at making cells more shear- and abrasion-sensitive, and therefore, more likely to be killed by the natural hydrodynamic forces of the circulatory system.     

The degradation of arsenobetaine to inorganic arsenic by sedimentary microorganisms

Two growth media containing arsenobetaine [(CH₃)₃ As⁺ CH₂COO^–] were mixed with coastal marine sediments, the latter providing a source of microorganisms. The mixtures were kept at 25 °C in the dark and shaken for several weeks under an atmosphere of air. The disappearance of arsenobetaine and the appearance of two metabolites were followed by HPLC. The HPLC-retention time of the first metabolite agreed with that of trimethylarsine oxide [(CH₃)₃AsO]. The second metabolite was identified as arsenate (As(V)) using hydride generation/cold trap/GC MS analysis and thin layer chromatography. This is the first scientific evidence showing that arsenobetaine is degraded by microorganisms to inorganic arsenic via trimethylarsine oxide. The degradation of arsenobetaine to inorganic arsenic completes the marine arsenic cycle that begins with the methylation of inorganic arsenic on the way to arsenobetaine.