cAMP IN THE CELL CYCLE OF THE DINOFLAGELLATE CRYPTHECODINIUM COHNII (DINOPHYTA)

The second messenger cAMP is a key regulator of growth in many cells. Previous studies showed that cAMP could reverse the growth inhibition of indoleamines in the dinoflagellate Crypthecodinium cohnii Biecheler. In the present study, we measured the level of intracellular cAMP during the cell cycle of C. cohnii . cAMP peaked during the G₁ phase and decreased to a minimum during S phase. Similarly, cAMP-dependent protein kinase activities peaked at both G₁ and G₂+M phases of the cell cycle, decreasing to a minimum at S phase. Addition of N6, O₂'-dibutyryl (Bt₂)-cAMP directly stimulated the growth of C. cohnii . Flow cytometric analysis of synchronized C. cohnii cells suggested that 1 mM cAMP shortened the cell cycle, probably at the exit from mitosis. The size of Bt₂-cAMP treated cells at G₁ was also larger than the control cells. The present study demonstrated a regulatory role of cAMP in the cell cycle progression in dinoflagellates.     

Evidence For the Presence of A Cdc2-Like Protein Kinase In the Dinoflagellate Crypthecodinium Cohnii

ABSTRACT. The unusual nature of mitosis and ancestral organization of the dinoflagellate nucleus prompted the question of whether the cdc 2-like histone H1 kinase, a presumed ubiquitous cell cycle regulator in eukaryotes, is present in these primitive organisms. Western blotting of Crypthecodinium cohnii protein extracts using antibody against the Pro-Ser-Thr-Ala-Ile-Arg-Glu (=PSTAIRE) amino acid sequence motif, conserved in all cdc 2 homologues known, revealed one prominent band corresponding to a protein with an apparent relative molecular weight ≈ 34,000, identical in mobility to that from HeLa cells and Physarum polycephalum , higher and lower eukaryotic controls, respectively. Incubation of C. cohnii cell lysates with p13 ^{suc 1}-sepharose beads, which preferentially, though not exclusively, bind p34 ^{cdc 2}, resulted in precipitation of a 34-kDa protein which was reactive with anti-PSTAIRE antibody, selectively competed for by the PSTAIRE peptide and able to phosphorylate histone H1 in vitro. We conclude that the dinoflagellate C. cohnii contains a protein very similar to the cdc 2 gene product from fission yeast and its homologues in all eukaryotes studied thus far.     

PCNA-LIKE PROTEINS IN DINOFLAGELLATES

Dinoflagellate chromosomes are permanently condensed and lack nucleosomes. These features suggest that dinoflagellate chromosomes must have an altered structural arrangement when compared to other eukaryotes and some modified DNA replication machinery to accommodate it. To investigate this possibility, proliferating cell nuclear antigen (PCNA), an essential component of the DNA replication machinery, was chosen for closer examination. A protein in the dinoflagellate Crypthecodinium cohnii Biecheler was found to react specifically with two monoclonal antibodies raised against PCNA. The observed band had a size of 55 kDa, which is far in excess of what has been described previously. Another dinoflagellate, Gymnodinium catenatum Bravo, also displayed a band of this size; however, a third species Amphidinium carterae Hulburt, had a band of lower molecular weight. The putative PCNA homolog in C. cohnii showed a nonconstitutive expression pattern. A time-course western blot using cells from a synchronized G₁ population showed that protein levels peak during S phase of the cell cycle. Both C. cohnii and A. carterae displayed a strong nuclear localization as determined by immunofluorescence microscopy. The signal was present in a subpopulation of cells, supporting a cell-cycle-specific expression pattern. It is possible that the larger size of this protein in some dinoflagellates reflects the unusual cell cycle and DNA arrangement of this group.     

Characterization of p80, a novel nuclear and cytoplasmic protein in dinoflagellates.

The presence of myosin in dinoflagellates was tested using an anti-Acanthamoeba castellanii myosin II polyclonal antibody on the heterotrophic dinoflagellate Crypthecodinium cohnii Seligo. Western blots revealed the presence of a unique band of 80 kDa in total protein extracts and after immunoprecipitation. Expression of this 80 kDa protein appeared constant during the different phases of the cell cycle. In protein extracts from various other dinoflagellates, this 80 kDa protein was detected only in the autotrophic species Prorocentrum micans Ehr. Screening of a C. cohnii cDNA expression library with this antibody revealed a cDNA coding for an amino acid sequence without homology in the databases. However, particular regions were detected: - a polyglutamine repeat domain in the N-terminal part of the protein, - four peptide sequences associated with GTP-binding sites, - a sequence with slight homology to the rod tail of Caenorhabditis elegans myosin II, -a sequence with homology to a human kinesin motor domain. Immunocytolocalization performed on C. cohnii thin sections with a polyclonal antibody raised against the recombinant protein showed p80 to be present both within the nucleus and in the cytoplasm. Labelling was widespread in the nucleoplasm and more concentrated at the periphery of the permanently condensed chromosomes. In the cytoplasm, labelling appeared in a punctate region close to the nucleus and in the flagellum. Potential functions of this novel protein are discussed.     

Predicting the elution behavior of proteins in affinity chromatography on non-porous particles.

Affinity chromatography on non-porous particles of microsize is particularly useful for the rapid analysis and micropreparative separation of proteins. The elution behavior of proteins in an affinity column packed with non-porous copolymerized particles of styrene, methyl methacrylate and glycidyl methacrylate was investigated both theoretically and experimentally, using the lysozyme-Cibacron Blue 3G-A affinity system. Equations used to predict the elution profiles, resulting from the elution by increasing the ionic strength (NaCl concentration) in the mobile phase, were obtained. The maximum adsorbate concentration, desorption rate constant and equilibrium constant under elution conditions were determined by matching experimental data with predicted elution profiles. Based on the parameters determined at a flow-rate of 0.5 ml/min and with 1 M NaCl in the elution buffer, the model equations could predict the elution profiles for other experimental runs, where different flow-rates and sodium chloride concentrations were used. Both the experimental and predicted results revealed that the affinity interaction kinetics are not significantly influenced by the flow-rate and, hence, the film mass transfer. To elute bound lysozyme from immobilized dye ligand, a higher value of the ionic strength leads to a faster elution and a sharper elution peak. The influence of elution conditions on the kinetic and thermodynamic parameters and, consequently, on the elution peak profiles was evaluated. The model equations can also predict the behavior of protein elution from an affinity column by changing the pH of the mobile phase, according to a previous study.     

Plasmids of<Emphasis Type="Italic">Staphylococcus cohnii</Emphasis> isolated from the intensive-care unit

Numerous isolates of both subspecies of Staphylococcus cohnii were found in the environment of the intensive-care unit of a pediatric hospital. These isolates carried in their cells many plasmids, up to fourteen, of a wide range of sizes (< 2 to > 56 kb). Striking was the occurrence of large plasmids not very common in staphylococci. These were present in > 80% of S. cohnii isolates. Fifty-two different plasmid profiles were found in 79 investigated isolates belonging to S. cohnii ssp. cohnii and S. cohnii ssp. urealyticus. Isolates similar in plasmid profiles were grouped in antibiotic-resistance clusters established for 9 antibiotics (gentamicin, ciprofloxacin, clindamycin, erythromycin, tetracycline, chloramphenicol, mupirocin, trimethoprim-sulfamethoxazole, vancomycin) using the method of unweighted pair group mathematical averages (UPGMA). Many isolates were multiresistant to antibiotics and produced bacteriocins.     

Large-surface biosensor technology for enhanced recovery in protein characterization.

A large-surface biosensor technique using surface plasmon resonance (SPR) was tested for protein purification by recovery of a monoclonal antibody against human proinsulin C-peptide. Notably, both reversible attachment/desorption and actual purification of the antibody from a multi-component protein mixture was shown. For initial chip attachment of the peptide ligand, C-peptide was biotinylated and attached to neutravidin on plastic chips with a large gold surface (effective area 26 mm(2)). Antibody binding and desorption was monitored in real-time SPR, and for elution different conditions were employed. Five percent formic acid (in contact with the chip surface for 3 min) in a 60-mul segment between air bubbles was efficient for subsequent analysis. In this manner, protein amounts up to 35 pmoles were recovered in a single capture/elution cycle. Evaluation by SDS-PAGE showed essentially no carryover between fractions in this elution process, and also not with other proteins in the mixture after purification. Compared to existing commercial instruments, this technique gives higher recovery and makes it possible to monitor monitor protein binding/desorption. Recovery of affinity partners at the multi-pmole level is demonstrated for protein purification in SPR approaches.     

An analyzer and monitor for rapid microscale peptide separations

An automatic peptide analyzer is described, which can be used to monitor quantitatively preparative microscale separations of protein hydrolysates, using microbore ion exchange columns in combination with volatile buffers. Complex protein mixtures can be effectively resolved within 5 h without deterioration of the elution profiles. By use of a novel system of split valves well-defined aliquots of the separated protein digest (5–25 nmol) are diverted into the monitor at regular intervals, while the rest of the eluate is collected intact for further characterization. Using low splitting ratios the peptide peaks become nearly indistinguishable from those obtained in analytical separations. At higher splitting ratios (1:5, 1:10) the sampling of early, rapidly eluting fractions becomes somewhat inaccurate. This is outweighed by the small amounts of material (less than 1 nmol) consumed by the analyzer under these conditions.     

Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell—free extracts of Xenopus laevis eggs

Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation,nuclear envelope assembly,and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle.The assembled nuclei,being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii.However,incubation of dinoflagellate Cyrthecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinium cohnii cells does not induce nuclear reconstitution.     

Periodicity and chaos in the response of Halobacterium to temporal light gradients

Halobacteria spontaneously reverse their swimming direction about every 10 s. This periodicity can be altered by light stimuli. We found that temporal exponential changes in light intensity, depending on wavelength and sign, lengthened or shortened the intervals between reversals. Within a limited range of steepness, light gradients enforced a new stable periodicity upon the system. Outside this range, they caused period doubling or induced a sequence of reversal events without any obvious regularity. An analysis of a functional relationship between apparently irregular periods by plotting each period as a function on the preceding one yielded a clearly discernible non-random structure, which shows some similarities to the one obtained by a model calculation for a periodically perturbed limit cycle oscillator. These results indicate that external forcing of the system may generate chaos. When the decay of intracellular sensory signals is delayed by inhibition of protein methylation the transition from periodic to aperiodic behavior occurs at a lower steepness of the gradient. We therefore assume that the generation of either periodic or deterministic chaotic behavior is determined by the relation between the signal lifetime and the frequency of stimulus inputs. The strong indications for transitions from periodic to chaotic behavior can be regarded as a further support of our hypothesis that the behavioral pattern of Halobacterium is controlled by an endogeneous oscillator.     

Changes in acid soluble nucleotide components of candida utilis during the cell cycle

Acid soluble extracts obtained at 30 min intervals from cells of C. utilis growing in synchrony in a phased culture (cycle time 51/2 hr) were fractionated on a Dowex-1-formate column. The series of fractionation profiles showed changes in number and amounts of components over the cell cycle. Transient accumulations of numerous components over the complex pool were observed. The significance of the changes are discussed in relation to practical applications and cell metabolism.     

The DNA of Volvox carteri: a biophysical and biosynthetic characterization.

Various biophysical and biosynthetic characteristics of deoxyribonucleic acid (DNA) from Volvox carteri are examined. The DNA from three strains (HK-10,NB-7 and KA-1) is compared, and all strains are shown to contain at least two distinct DNA species which band at densities of 1.714-1.715 and 1.704-1.705 g/cm3 in neutral CsCl and correspond to nuclear and "cytoplasmic" DNA, respectively. Base compositions calculated from these densities, 55-56% G+C for nuclear DNA, and 45-46% G+C for cytoplasmic DNA, are in close agreement with % G+C values estimated from thermal denaturation data. DNA from strain KA-1 has a third component with a buoyant density of 1.693 g/cm3. DNA synthesis is analysed using radioactively labelled heterogeneously grown strains of Volvox carteri and profiles obtained following preparative CsCl density gradient centrifugation are presented. In addition, dissimilarities in patterns of DNA synthesis at various periods in the asexual life cycle are reported for synchronous cultures of strain HK-10. These differences in temporal patterns of DNA synthesis clearly indicate that while nuclear DNA is make to some degree throughout the life cycle, cytoplasmic DNA synthesis appears to occur only at discrete intervals.     

Investigation on Stability of Transporter Protein, Glucuronide Transporter from Escherichia coli

The glucuronide transporter GusB, the product of the gusB gene from Escherichia coli, is responsible for detoxification of metabolites. In this study, we successfully expressed GusB homologously in E. coli and investigated its oligomeric state in n-dodecyl-β-d-maltoside (DDM) detergent solution. Evidence for a pentameric state with a Stokes radius of 57 ± 2 Å for the purified GusB protein in DDM solution was obtained by analytical size-exclusion HPLC. The elution peak corresponding to pentameric GusB is commonly seen in elution profiles in the different buffer systems examined over a wide pH range. Hence, it is likely that GusB resides in the membrane as a pentamer. Stability studies with different incubation periods with the typical lipids, such as dimyristoylphosphatidylcholine, and total E. coli phospholipids, as the representatives of both phosphatidylcholine and phosphatidylethanolamine, show some clues to two-dimensional crystallization of GusB with lipids.     

Synthesis of glycoprotein, glycolipid, protein, and lipid in synchronized L5178Y cells

Synthesis of four macromolecular classes found in membranes—glycoprotein, glycolipid, protein, and lipid—was measured as a function of time of the cell cycle in synchronized L5178Y cells. Incorporation of leucine, choline, fucose, glucosamine, or thymidine into the cells, protein, nucleic acid, or lipid was measured by pulse-labeling for ½ hr at ½ hr intervals after release from the mitotic block. The amount of protein, lipid, glycoprotein, or glycolipid released or secreted into the medium by the L5178Y cells was also measured as a function of time of the cell cycle. Cellular protein was found to be synthesized throughout the cell cycle, with the highest synthesis occurring in the S period; synthesis was depressed in the M period. Cellular glycoprotein was synthesized at approximately the same times as protein, except that the rates of glycoprotein synthesis in the S period relative to other periods were much greater than for protein. Secreted protein was synthesized throughout the cell cycle without any general pattern, except that secretion was elevated in the late S and G₂ periods. Secreted glycoprotein was similar to secreted protein. Cellular lipid and cellular glycolipid were synthesized almost exclusively in the G₂ and M periods; there was no synthesis in the G₁ and S periods. Release or secretion of glycolipid and lipid also occurred in the G₂ and M periods.     

Restricted diffusion effect on the binding of proteins to porous polymer resins as studied by repetitive injection method

A solution of bovine serum albumin (BSA) is repeatedly injected into a column packed with highly porous and hydrophobic polymer resins at appropriate intervals. The injected BSA is thoroughly retained in the column for 10 injections and, afterwards, starts to be eluted from the column gradually. Taking into consideration the restricting effect of already bound BSA upon the diffusion of newly injected BSA into the pores of the polymer resins, we can interpret the BSA elution profile from columns packed with polymer resin of various pore sizes and porosities. The effects of the binding rate constant and BSA concentration upon the elution profiles of BSA are also analyzed. Formyl groups are introduced into the polymers as a binding site with proteins, and the elution profiles of BSA from the column packed with the formylated resin are also analyzed.     

Inhibition of cell proliferation by mechanical agitation involves transient cell cycle arrest at G1 phase in dinoflagellates

Summary.  Cell proliferation of dinoflagellates is negatively affected by mechanical agitation and red tides caused by members of the group have been correlated with periods of calm sea conditions. The mechanism involved in the mechanically transduced inhibition of cell proliferation is thought to involve the disruption of the cell division apparatus. In this study, we used highly synchronized cells and flow cytometry to study the effects of mechanical agitation on cell cycle progression. We observed that mechanical agitation induced transient cell cycle arrest at G₁ phase, in both the heterotrophic dinoflagellate Crypthecodinium cohnii and the photosynthetic dinoflagellate Heteroscapsa triquetra. Received March 12, 2002; accepted July 20, 2002; published online November 29, 2002     

Interactions between extracellular matrix components and proteoglycans released from monocytesin vitro

³⁵S-labelled chondroitin sulfate proteoglycans isolated from conditioned media of cultured human monocytes (day 1in vitro) and monocyte-derived macrophages (day 6in vitro) were chromatographed on columns of immobilized fibronectin and collagen, respectively. The elution profiles prior to and after alkali treatment were compared with those of standards chondroitin 4-sulfate and chondroitin sulfate E and heparin. The day 6³⁵S-proteoglycans have a higher sulfate density than the day 1 species, but this difference did not affect the elution profiles after chromatography on collagen-Sepharose, whereas the day 6 proteoglycans bound more firmly than the day 1 fraction to fibronectin-Sepharose. The elution patterns obtained for these distinct proteoglycans closely resembled those of heparin and oversulfated chondroitin sulfate E standards, and clearly demonstrated the importance of sulfate density both for the affinity to fibronectin and collagen. Neither day 1 nor day 6³⁵S-proteoglycans were found to interact with hyaluronate.Abbreviations used CSPG chondroitin sulfate proteoglycan - GAG glycosaminoglycan - CS chondroitin sulfate - CS-E chondroitin 4,6 disulfate - MDM monocyte-derived macrophages     

Lipid biosynthesis and its coordination with cell cycle progression

The activation of cell cycle regulators at the G1/S boundary has been linked to the cellular protein synthesis rate. It is conceivable that regulatory mechanisms are required to allow cells to coordinate the synthesis of other macromolecules with cell cycle progression. The availability of highly synchronized cells and flow cytometric methods facilitates investigation of the dynamics of lipid synthesis in the entire cell cycle of the heterotrophic dinoflagellate Crypthecodinium cohnii. Flow cytograms of Nile red-stained cells revealed a stepwise increase in the polar lipid content and a continuous increase in neutral lipid content in the dinoflagellate cell cycle. A cell cycle delay at early G1, but not G2/M, was observed upon inhibition of lipid synthesis. However, lipid synthesis continued during cell cycle arrest at the G1/S transition. A cell cycle delay was not observed when inhibitors of cellulose synthesis and fatty acid synthesis were added after the late G1 phase of the cell cycle. This implicates a commitment point that monitors the synthesis of fatty acids at the late G1 phase of the dinoflagellate cell cycle. Reduction of the glucose concentration in the medium down-regulated the G1 cell size with a concomitant forward shift of the commitment point. Inhibition of lipid synthesis up-regulated cellulose synthesis and resulted in an increase in cellulosic contents, while an inhibition of cellulose synthesis had no effects on lipid synthesis. Fatty acid synthesis and cellulose synthesis are apparently coupled to the cell cycle via independent pathways.