Chemical acetylation, trypsinolysis and stability of nucleosomes

Gel electrophoretic analysis of the histone chemical acetylation in the nucleosome core particles with acetic andydride revealed availability of about 14 lysine residues of histone H2A, 15-21 of H2B, 8-11--H3 and 6-9--H4. Moderately lysine-rich histones H2A and H2B were found to be more susceptible to acetylation than arginine-rich H3 and H4. Chemical acetylation enhanced the rate of trypsin digestion in acetylated nucleosomes as evidenced by gel electrophoresis of histone fragments. A more pronounced trypsin digestion was evident at acetylation of only 3-5 histone amino groups per nucleosome. However, even heavily acetylated nucleosomes yielded in familiar trypsin limit digest pattern of histone fragments thus indicating persistence of histone octamer. Nucleosomes which were trace acetylated (up to 3-5 histone amino groups neutralized per nucleosome) and treated with trypsin to remove highly charged terminal histone regions revealed remarkable unfolding and partial dissociation when analyzed by gel electrophoresis. The same trace acetylated nucleosomes did not show such destabilization prior to trypsin digestion.     

The spatial position and replication timing of chromosomal domains are both established in early G1 phase

Mammalian chromosomal domains replicate at defined, developmentally regulated times during S phase. The positions of these domains in Chinese hamster nuclei were established within 1 hr after nuclear envelope formation and maintained thereafter. When G1 phase nuclei were incubated in Xenopus egg extracts, domains were replicated in the proper temporal order with nuclei isolated after spatial repositioning, but not with nuclei isolated prior to repositioning. Mcm2 was bound both to early- and late-replicating chromatin domains prior to this transition whereas specification of the dihydrofolate reductase replication origin took place several hours thereafter. These results identify an early G1 phase point at which replication timing is determined and demonstrate a provocative temporal coincidence between the establishment of nuclear position and replication timing.     

Self-aggregation--an intrinsic property of G(M1) in lipid bilayers

We demonstrate that the ganglioside G(M1) in lipid bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) exhibits a non-uniform lateral distribution, i.e., enriched regions of GM(1) molecules are formed, which is an argument in favour of self-aggregation of G(M1) being an intrinsic property of G(M1) ganglioside. This was concluded from energy transfer/migration studies of BODIPY-labelled gangliosides by means of time-resolved fluorescence lifetime and depolarization experiments. Three fluorophore-labelled gangliosides were synthesized to include either of two spectroscopically different BODIPY groups. These were specifically localized either in the polar headgroup region or in the non-polar region of the lipid bilayer. An eventual ganglioside-ganglioside affinity/aggregation induced by the BODIPY groups was experimentally excluded, which suggests their use in examining the influence of G(M1) in more complex systems.     

Regulated expression of cofilin and the consequent regulation of p27kip1 are essential for G1 phase progression

Cofilin, a ubiquitously expressed actin binding protein, is responsible for the formation of the actin cytoskeleton and is indispensable for cell cycle control. However, the association between cofilin expression and the cell cycle remains to be elucidated. In this study, we found that the expression level of cofilin up-regulated in G1 phase-arrested confluent cells, while knockdown of cofilin expression by small interference RNA (siRNA) in these cells led to a reduction in the population of G1 cells. To investigate the role of cofilin in the control of G1 phase progression, a tet-on gene expression system was introduced to over-express different concentrations of cofilin in cells. The results showed that G1 phase progression was blocked following induction of exogenous cofilin. A survey of the cell cycle proteins controlling the G1 phase progression revealed that the cyclin-dependent kinase inhibitor (CKI) p27kip1 was the primary molecule induced by over-expressed cofilin in a time and dose dependent manner. Up-regulated p27kip1 repressed phosphorylation of the retinoblastoma protein (Rb) mediated by cyclin D1/CDK4 activity. Conversely, siRNA against p27kip1 expression in the cofilin over-expressing cells released the G1 phase arrest. Furthermore, we found that over-expression of cofilin led to induction of p27kip1 gene promoter transactivation using luciferase reporter gene assay. This effect was associated with increase of p27kip1 mRNA transiently. In addition, inhibition of threonine-187 phosphorylation of p27kip1 protein for ubiquitinyl-proteasomal mediated degradation was also involved in up-regulation of p27 kip1. These data suggest that cofilin expression and its regulation of p27kip1 expression is important for the control of G1 phase progression.     

Conserved and unique features of the fission yeast core Atg1 complex

Although the human ULK complex mediates phagophore initiation similar to the budding yeast Saccharomyces cerevisiae Atg1 complex, this complex contains ATG101 but not Atg29 and Atg31. Here, we analyzed the fission yeast Schizosaccharomyces pombe Atg1 complex, which has a subunit composition that resembles the human ULK complex. Our pairwise coprecipitation experiments showed that while the interactions between Atg1, Atg13, and Atg17 are conserved, Atg101 does not bind Atg17. Instead, Atg101 interacts with the HORMA domain of Atg13 and this enhances the stability of both proteins. We also found that S. pombe Atg17, the putative scaffold subunit, adopts a rod-shaped structure with no discernible curvature. Interestingly, S. pombe Atg17 binds S. cerevisiae Atg13, Atg29, and Atg31 in vitro, but it cannot complement the function of S. cerevisiae Atg17 in vivo. Furthermore, S. pombe Atg101 cannot substitute for the function of S. cerevisiae Atg29 and Atg31 in vivo. Collectively, our work generates new insights into the subunit organization and structural properties of an Atg101-containing Atg1/ULK complex.     

Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast

The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A(Cnp1) with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A(Cnp1) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle.     

Identifiability and retrievability of unique parameters describing intrinsic Andrews kinetics

A key factor contributing to the variability in the microbial kinetic parameters reported from batch assays is parameter identifiability, i.e., the ability of the mathematical routine used for parameter estimation to provide unique estimates of the individual parameter values. This work encompassed a three-part evaluation of the parameter identifiability of intrinsic kinetic parameters describing the Andrews growth model that are obtained from batch assays. First, a parameter identifiability analysis was conducted by visually inspecting the sensitivity equations for the Andrews growth model. Second, the practical retrievability of the parameters in the presence of experimental error was evaluated for the parameter estimation routine used. Third, the results of these analyses were tested using an example data set from the literature for a self-inhibitory substrate. The general trends from these analyses were consistent and indicated that it is very difficult, if not impossible, to simultaneously obtain a unique set of estimates of intrinsic kinetic parameters for the Andrews growth model using data from a single batch experiment.     

Shared and unique features of evolutionary diversification

A fundamental question in evolutionary biology asks whether organisms experiencing similar selective pressures will evolve similar solutions or whether historical contingencies dominate the evolutionary process and yield disparate evolutionary outcomes. It is perhaps most likely that both shared selective forces as well as unique histories play key roles in the course of evolution. Consequently, when multiple species face a common environmental gradient, their patterns of divergence might exhibit both shared and unique elements. Here we describe a general framework for investigating and evaluating the relative importance of these contrasting features of diversification. We examined morphological diversification in three species of livebearing fishes across a predation gradient. All species (Gambusia affinis from the United States of America, Brachyrhaphis rhabdophora from Costa Rica, and Poecilia reticulata from Trinidad) exhibited more elongate bodies, a larger caudal peduncle, and a relatively lower position of the eye in predator populations. This shared response suggests that common selective pressures generated parallel outcomes within three different species. However, each species also exhibited unique features of divergence, which might reflect phylogenetic tendencies, chance events, or localized environmental differences. In this system, we found that shared aspects of divergence were of larger magnitude than unique elements, suggesting common natural selective forces have played a greater role than unique histories in producing the observed patterns of morphological diversification. Assessing the nature and relative importance of shared and unique responses should aid in elucidating the relative generality or peculiarity in evolutionary divergence.     

Length of human prematurely condensed chromosomes during G0 and G1 phase

Length measurements on C-banded prematurely condensed no. 1 human chromosomes of G0 and G1 lymphocytes, as well as of synchronized G1 HEp cells revealed that (i) no length difference exists between mitotic chromosomes and G0 chromosomes; (ii) 1 h after PHA stimulation a clear increase in length is detectable; (iii) in isolated cases an increase by the factor 5 can be observed during G1; (iv) the increase is significantly less for constitutive heterochromatin than for euchromatin. The possibility is discussed that these conformational changes of chromatin reflect physiological differences, i.e. the rate of RNA synthesis during interphase.     

Assembly and stability of nisin-lipid II pores

The peptide antibiotic nisin was the first reported example of an antibiotic that kills bacteria via targeted pore formation. The specific target of nisin is Lipid II, an essential intermediate in the bacterial cell-wall synthesis. High-affinity binding of the antibiotic to Lipid II is followed by rapid permeabilization of the membrane. Here, we investigated the assembly and stability of nisin-Lipid II pore complexes by means of pyrene fluorescence and circular dichroism. We demonstrated that nisin uses all available Lipid II molecules in the membrane to form pore complexes. The pore complexes have a uniform structure and consist of 8 nisin and 4 Lipid II molecules. Moreover, the pores displayed a remarkable stability, because they were able to resist the solubilization of the membrane environment by mild detergents. Similar experiments with [N20P/M21P]nisin showed that the hinge region is essential for the assembly into stable pore complexes. The new insights were used to propose a refined model for nisin pore formation.     

Assembly and trafficking of heterotrimeric G proteins

To be activated by cell surface G protein-coupled receptors, heterotrimeric G proteins must localize at the cytoplasmic surface of plasma membranes. Moreover, some G protein subunits are able to traffic reversibly from the plasma membrane to intracellular locations upon activation. This current topic will highlight new insights into how nascent G protein subunits are assembled and how they arrive at plasma membranes. In addition, recent reports have increased our knowledge of activation-induced trafficking of G proteins. Understanding G protein assembly and trafficking will lead to a greater understanding of novel ways that cells regulate G protein signaling.     

Acidic stress induced G1 cell cycle arrest and intrinsic apoptotic pathway in Jurkat T-lymphocytes

Background

Low extracellular pH (pH_e) is a common hallmark of tumor microenvironment, which will also affect pH sensitive T-lymphocytes in this environment. Due to the growing interest on T-cell mediated cancer therapies, acidic stress induced consequences on this lymphocyte deserves through investigations.

Messenger RNAs coding for enzymes of polyamine biosynthesis are induced during the G0-G1 transition but not during traverse of the normal G1 phase

The events occurring during emergence of cells from quiescence (G0) are not necessarily identical to those in the G1 phase of continuously dividing cells. Cellular levels of the mRNAs coding for ornithine decarboxylase (ODC) and S-adenosyl-methionine decarboxylase (SDC), key enzymes in polyamine synthesis, increased maximally within 5 h after addition of serum to resting 3T3 cells, following a kinetic course similar to that of c-myc mRNA. In a pure early G1 population of cells, prepared by centrifugal elutriation of growing fibroblasts, the levels of ODC and SDC mRNAs were not significantly lower than in other phases of the cell cycle and approximated serum-induced levels rather than the reduced values found in serum-starved cells. Thus, we conclude that the mRNAs coding for the polyamine biosynthetic enzymes, like c-myc, are growth controlled, but not regulated during traverse of a normal cell cycle.     

Aflatoxins B1 and G1 solubility in standard solutions and stability during cold storage

In the present work we study the use of different solvents to store aflatoxins B₁ and G₁ standard solutions. We have obtained significant differences between aflatoxin B₁ and G₁ In ethyl acetate, methanol and water, with aflatoxin G₁ being less stable. We recommend chloroform as the election solvent to store the aflatoxin solutions. The fact that aflatoxins are highly stable in water may have a potential use in experiments of biological activity.