Cyclic AMP-dependent and -independent protein phosphokinase activity in subcellular fractions of synchronously growing leydig I-10 cells.

The Leydig I-10 tumor cell line was synchronized by the double thymidine block method using 1.0 mM thymidine. Protein phosphokinase activity of subcellular fractions was determined at various times throughout the cell cycle. Microsomal cAMP-independent kinase activity increased in G2 and decreased during the S and G1 phases. Except for relatively small increases during the G1 and late S phases, microsomal cAMP-dependent kinase activity remained unchanged throughout most of the cycle. In the lysosomal-mitochondrial fraction, cAMP-dependent and cAMP-independent protein kinase activity increased during the S phase. Independent kinase activity peaked again during G1, while the dependent kinase became depressed. Phosphokinase activity increased in the nuclear fraction in late G2 and during mitosis, and was due to increases in both cAMP-independent and cAMP-dependent kinase activity. Cytosol cAMP-dependent kinase activity increased in G2 and during mitosis; cAMP-independent kinase activity showed some increased activity during late G2 and mitosis. These temporal variations in the subcellular kinase activities throughout the cell cycle may act to phosphorylate subcellular protein substrates in a cell cycle-specific fashion.     

Molecular mechanisms creating bistable switches at cell cycle transitions

Progression through the eukaryotic cell cycle is characterized by specific transitions, where cells move irreversibly from stage i−1 of the cycle into stage i. These irreversible cell cycle transitions are regulated by underlying bistable switches, which share some common features. An inhibitory protein stalls progression, and an activatory protein promotes progression. The inhibitor and activator are locked in a double-negative feedback loop, creating a one-way toggle switch that guarantees an irreversible commitment to move forward through the cell cycle, and it opposes regression from stage i to stage i−1. In many cases, the activator is an enzyme that modifies the inhibitor in multiple steps, whereas the hypo-modified inhibitor binds strongly to the activator and resists its enzymatic activity. These interactions are the basis of a reaction motif that provides a simple and generic account of many characteristic properties of cell cycle transitions. To demonstrate this assertion, we apply the motif in detail to the G1/S transition in budding yeast and to the mitotic checkpoint in mammalian cells. Variations of the motif might support irreversible cellular decision-making in other contexts.     

Who guards the guardian? Mechanisms that restrain APC/C during the cell cycle

The cell cycle is principally controlled by Cyclin Dependent Kinases (CDKs), whose oscillating activities are determined by binding to Cyclin coactivators. Cyclins exhibit dynamic changes in abundance as cells pass through the cell cycle. The sequential, timed accumulation and degradation of Cyclins, as well as many other proteins, imposes order on the cell cycle and contributes to genome maintenance. The destruction of many cell cycle regulated proteins, including Cyclins A and B, is controlled by a large, multi-subunit E3 ubiquitin ligase termed the Anaphase Promoting Complex/Cyclosome (APC/C). APC/C activity is tightly regulated during the cell cycle. Its activation state increases dramatically in mid-mitosis and it remains active until the end of G1 phase. Following its mandatory inactivation at the G1/S boundary, APC/C activity remains low until the subsequent mitosis. Due to its role in guarding against the inappropriate or untimely accumulation of Cyclins, the APC/C is a core component of the cell cycle oscillator. In addition to the regulation of Cyclins, APC/C controls the degradation of many other substrates. Therefore, it is vital that the activity of APC/C itself be tightly guarded. The APC/C is most well studied for its role and regulation during mitosis. However, the APC/C also plays a similarly important and conserved role in the maintenance of G1 phase. Here we review the diverse mechanisms counteracting APC/C activity throughout the cell cycle and the importance of their coordinated actions on cell growth, proliferation, and disease.     

The metabolic switch and its regulation in cancer cells

The primary features of cancer are maintained via intrinsically modified metabolic activity, which is characterized by enhanced nutrient supply, energy production, and biosynthetic activity to synthesize a variety of macromolecular components during each passage through the cell cycle. This metabolic shift in transformed cells, as compared with non-proliferating cells, involves aberrant activation of aerobic glycolysis, de novo lipid biosynthesis and glutamine-dependent anaplerosis to fuel robust cell growth and proliferation. Here, we discuss the unique metabolic characteristics of cancer, the constitutive regulation of metabolism through a variety of signal transduction pathways and/or enzymes involved in metabolic reprogramming in cancer cells, and their implications in cancer diagnosis and therapy.     

NMR analysis of a cell division cycle mutant of Saccharomyces cerevisiae

cdc 19.1 is a temperature-sensitive lesion in the genome of Saccharomyces cerevisiae. The phenotype of this mutant is a cell cycle specific arrest in G₁, which is expressed at 37°C. In the present study, ³¹P- and ¹³C-NMR spectroscopy were used to analyze the metabolism of the mutant at the permissive and restrictive temperatures. Our results confirm previous findings which have indicated that cdc 19.1 contains temperature-sensitive pyruvate kinase activity. In contrast to previous findings, however, the present investigation demonstrates that restriction of pyruvate kinase activity in vivo takes as long as 24 h to be fully expressed. In addition, analysis by NMR has allowed us to assess the metabolic consequences of pyruvate kinase restriction which may contribute to the arrest of cell growth in the early G₁ phase of the cell division cycle.     

Analysis of metabolic flux in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing human-like collagen in fed-batch culture

Metabolic flux distributions of recombinant Escherichia coli BL21 expressing human-like collagen were determined by means of a stoichiometric network and metabolic balancing. At the batch growth stage, the fluxes of the pentose phosphate pathway were higher than the fluxes of the fed-batch growth phase and the production stage. After the temperature was increased, there was a substantially elevated energy demand for synthesizing human-like collagen and heat-shock proteins, which resulted in changes in metabolic fluxes. The activities of the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cycle were significantly enhanced, leading to a reduction in the fluxes of the pentose phosphate pathway and other anabolic pathways. The temperature upshift also caused an increase in NADPH production by isocitrate dehydrogenase in the tricarboxylic acid cycle. The metabolic model predicted the involvement of a transhydrogenase that generates additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. These data indicated that the maintenance energy for cellular activity increased with the increase in biomass in fed-batch culture, and that cell growth and synthesis of human-like collagen could clearly represent the changes in metabolic fluxes. At the production stage, more NADPH was used to synthesize human-like collagen than for maintaining cellular activity, cell growth, and cell propagation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Increase of Mitochondrial Fraction in Sweet Potato Root Tissue after Wounding or Infection with Ceratocystis fimbriata

The acid-insoluble nitrogen content, lipid content, and cytochrome oxidase activity in the mitochondrial fraction are found to increase during incubation of slices of sweet potato (Ipomoea batatas) root tissue. These increases appear to be related to an increase in the number of the mitochondrial particles. The increase in the mitochondrial fraction is not accompanied by an increase in cell number. The nitrogen content in the mitochondrial fraction increases prior to the changes in the activity of cytochrome oxidase and lipid content. The increase in the numbers of the mitochondrial particles lags behind the increase in the cytochrome oxidase activity. Such findings are also found in the tissue infected by Ceratocystis fimbriata.     

The mechanism of regulation of fibroblastic cell replication. III. A central role for nutrient limitation: a conditioning effect due to serum imprinting of the cell response

Haploid Saccharomyces cerevisiae cells of mating type a, but not α, produce and secrete a diffusible substance, designated a factor. The a factor transiently arrests cells of mating type α, but not a, at a very early stage of the cell cycle, prior to budding and to the initiation of DNA synthesis. While the cells are arrested at this stage, few, if any, of the functions required for the ensuing cell cycle are carried out. This stage of the cell cycle coincides with the stage at which α factor, produced by cells of mating type a, specifically arrests cells of mating type a [2]. It seems probable that the reciprocally acting a and α factors together provide the mechanism by which haploid cells are synchronized to the appropriate stage of the cell cycle as a prelude to conjugation.     

Nuclear Pore Complex Number and Distribution throughout the Saccharomyces cerevisiae Cell Cycle by Three-Dimensional Reconstruction from Electron Micrographs of Nuclear Envelopes

The number of nuclear pore complexes (NPCs) in individual nuclei of the yeast Saccharomyces cerevisiae was determined by computer-aided reconstruction of entire nuclei from electron micrographs of serially sectioned cells. Nuclei of 32 haploid cells at various points in the cell cycle were modeled and found to contain between 65 and 182 NPCs. Morphological markers, such as cell shape and nuclear shape, were used to determine the cell cycle stage of the cell being examined. NPC number was correlated with cell cycle stage to reveal that the number of NPCs increases steadily, beginning in G₁-phase, suggesting that NPC assembly occurs continuously throughout the cell cycle. However, the accumulation of nuclear envelope observed during the cell cycle, indicated by nuclear surface area, is not continuous at the same rate, such that the density of NPCs per unit area of nuclear envelope peaks in apparent S-phase cells. Analysis of the nuclear envelope reconstructions also revealed no preferred NPC-to-NPC distance. However, NPCs were found in large clusters over regions of the nuclear envelope. Interestingly, clusters of NPCs were most pronounced in early mitotic nuclei and were found to be associated with the spindle pole bodies, but the functional significance of this association is unknown.     

Immunomodulation-mediated anticancer activity of a novel compound from Brugmansia suaveolens leaves

Immunomodulation activity-guided fractionation of ethanol extract of Brugmansia suaveolens leaves was carried out to isolate a novel compound SUPH036-022A (1) by co-culturing the test fraction/compound activated PBMC with MCF7 and A549 cancer cell lines. Assessment of immune markers in PBMC, and analysis of apoptosis markers and cell cycle was carried out for cancer cells. The structure of the isolated compound was elucidated by spectral analysis. Compound 1 enhanced the secretion of immune markers, IL-2 and IFN-γ, from PBMC. Further, compound 1 treated PBMC increased cell death in MCF7 and A549 cell lines and induced ROS production and mitochondrial membrane perturbation, leading to apoptosis. Flow cytometry analysis revealed; compound 1 stimulated PBMC to cause a five-fold increase in cell cycle perturbations in the sub-G1 stage of cancer cells as compared to the negative control. The compound, in the absence of PBMC, only had a weak cytotoxic activity against these cell lines. Thus, compound 1 is a novel lead for immunomodulation-mediated anticancer activity.     

The cell cycle and its relationship to development in Dictyostelium discoideum.

When deprived of exogenous nutrients some amoebas of Dictyostelium discoideum do continue to progress through the cell cycle. There are two distinct periods when mitotic cell division occurs. Labeling studies show that during the first period, which begins at the onset of development and ceases at the first visible signs of aggregation (rippling), only those cells which are beyond a certain point in G2 at the initiation of development divide. The second period of mitotic activity begins at tip formation, reaches maximum activity at the grex stage, and ceases during early culmination. Significantly, examination of the development of amoebas harvested when in the stationary phase of growth (and thus arrested in G2) shows that these cells still undergo mitotic cell division during the second period but do not show any such division during the preaggregation phase. The extent to which increases in cell number can be taken to be indicative of mitotic cell division varies from one culture to another due to the presence of variable numbers of multinucleate cells which become mononucleate during the first 10 hr of development. However, when due allowance has been made for the existence of these cells in axenically growing amoebal populations, our data show that by completion of fruiting body construction there has been a doubling in cell number as a direct result of mitotic cell division. Nuclear DNA synthesis also occurs at two distinct periods during development, these coinciding with the periods of mitotic activity. However, since no more than 35% of the cells have undergone nuclear DNA synthesis by the end of the developmental phase, our results are inconsistent with the conclusion that all cells accumulate at a position in G2 at the time of aggregation. Our results do suggest, however, that mitotic cell division of a fraction of the cells may be an integral part of the developmental phase.     

Mechanochemical regulation of oscillatory follicle cell dynamics in the developing Drosophila egg chamber

During tissue elongation from stage 9 to stage 10 in Drosophila oogenesis, the egg chamber increases in length by ∼1.7-fold while increasing in volume by eightfold. During these stages, spontaneous oscillations in the contraction of cell basal surfaces develop in a subset of follicle cells. This patterned activity is required for elongation of the egg chamber; however, the mechanisms generating the spatiotemporal pattern have been unclear. Here we use a combination of quantitative modeling and experimental perturbation to show that mechanochemical interactions are sufficient to generate oscillations of myosin contractile activity in the observed spatiotemporal pattern. We propose that follicle cells in the epithelial layer contract against pressure in the expanding egg chamber. As tension in the epithelial layer increases, Rho kinase signaling activates myosin assembly and contraction. The activation process is cooperative, leading to a limit cycle in the myosin dynamics. Our model produces asynchronous oscillations in follicle cell area and myosin content, consistent with experimental observations. In addition, we test the prediction that removal of the basal lamina will increase the average oscillation period. The model demonstrates that in principle, mechanochemical interactions are sufficient to drive patterning and morphogenesis, independent of patterned gene expression.     

Macrophage reaction in the bed of an allogeneic skin transplant]

The character of cell reactions, number of macrophages at the place of skin allograft was demonstrated to depend on the stage of posttransplantational period. Macrophagal reaction increases as the crisis of rejection is approaching; functional and metabolic activity of macrophages is increasing that is evident from an intensive vacuolization of cytoplasm, decreased contents of glycogene, enhanced acid phosphatase activity in them, increased amount of H3-thymidin labelled cells.     

Tomato Fruit Cell Wall Synthesis during Development and Senescence : In Vivo Radiolabeling of Wall Fractions Using [C]Sucrose

The pedicel of tomato fruit (Lycopersicon esculentum Mill., cv `Rutgers') of different developmental stages from immature-green (IG) to red was injected on the vine with 7 microcuries [¹⁴C(U)]sucrose and harvested after 18 hours. Cell walls were isolated from outer pericarp and further fractionated yielding ionically associated pectin, covalently bound pectin, hemicellulosic fraction I, hemicellulosic fraction II, and cellulosic fraction II. The dry weight of the total cell wall and of each cell wall fraction per gram fresh weight of pericarp tissue decreased after the mature-green (MG) stage of development. Incorporation of radiolabeled sugars into each fraction decreased from the IG to MG3 (locules jellied but still green) stage. Incorporation in all fractions increased from MG3 to breaker and turning (T) and then decreased from T to red. Data indicate that cell wall synthesis continues throughout ripening and increases transiently from MG4 (locules jellied and yellow to pink in color) to T, corresponding to the peak in respiration and ethylene synthesis during the climacteric. Synthesis continued at a time when total cell wall fraction dry weight decreased indicating the occurrence of cell wall turnover. Synthesis and insertion of a modified polymer with removal of other polymers may produce a less rigid cell wall and allow softening of the tissue integrity during ripening.