Wild-type p53-induced phosphatase 1 (Wip1) forestalls cellular premature senescence at physiological oxygen levels by regulating DNA damage response signaling during DNA replication

Wip1 (protein phosphatase Mg²⁺/Mn²⁺-dependent 1D, Ppm1d) is a nuclear serine/threonine protein phosphatase that is induced by p53 following the activation of DNA damage response (DDR) signaling. Ppm1d^−/− mouse embryonic fibroblasts (MEFs) exhibit premature senescence under conventional culture conditions; however, little is known regarding the role of Wip1 in regulating cellular senescence. In this study, we found that even at a representative physiological concentration of 3% O₂, Ppm1d^−/− MEFs underwent premature cellular senescence that depended on the functional activation of p53. Interestingly, Ppm1d^−/− MEFs showed increased H2AX phosphorylation levels without increased levels of reactive oxygen species (ROS) or DNA base damage compared with wild-type (Wt) MEFs, suggesting a decreased threshold for DDR activation or sustained DDR activation during recovery. Notably, the increased H2AX phosphorylation levels observed in Ppm1d^−/− MEFs were primarily associated with S-phase cells and predominantly dependent on the activation of ATM. Moreover, these same phenotypes were observed when Wt and Ppm1d^−/− MEFs were either transiently or chronically exposed to low levels of agents that induce replication-mediated double-stranded breaks. These findings suggest that Wip1 prevents the induction of cellular senescence at physiological oxygen levels by attenuating DDR signaling in response to endogenous double-stranded breaks that form during DNA replication.     

Effect of overexpression of β- and γ-actin isoforms on actin cytoskeleton organization and migration of human colon cancer cells

Actins are eukaryotic proteins, which are involved in diverse cellular functions including muscle contraction, cell motility, adhesion and maintenance of cell shape. Cytoplasmic actin isoforms β and γ are ubiquitously expressed and essential for cell functioning. However, their unique contributions are not very well understood. The aim of this study was to determine the effect of β- and γ-actin overexpression on the migration capacity and actin cytoskeleton organization of human colon adenocarcinoma BE cells. In cells overexpressing β- or γ-actin, distinct cytoskeletal actin rearrangements were observed under the laser scanning confocal microscope. Overexpressed actins localized at the submembranous region of the cell body, especially near to the leading edge and on the tips of pseudopodia. The cells transfected with plasmids containing cDNA for β- or γ-actin were characterized by increased migration and invasion capacities. However, the migration velocity was statistically significantly higher only in the case of γ-actin overexpressing cells. In conclusion, the increased level of β- or γ-actin leads to actin cytoskeletal remodeling followed by an increase in migration and invasion capacities of human colon BE cells. These data suggest that expression of both actin isoforms has an impact on cancer cell motility, with the subtle predominance of γ-actin, and may influence invasiveness of human colon cancer.     

Insights into the structural nature of the transition state in the Kir channel gating pathway

In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a ‘catalytic’ effect upon the channel gating kinetics.     

Ultra-rapid warming yields high survival of mouse oocytes cooled to -196°c in dilutions of a standard vitrification solution

Intracellular ice is generally lethal. One way to avoid it is to vitrify cells; that is, to convert cell water to a glass rather than to ice. The belief has been that this requires both the cooling rate and the concentration of glass-inducing solutes be very high. But high solute concentrations can themselves be damaging. However, the findings we report here on the vitrification of mouse oocytes are not in accord with the first belief that cooling needs to be extremely rapid. The important requirement is that the warming rate be extremely high. We subjected mouse oocytes in the vitrification solution EAFS 10/10 to vitrification procedures using a broad range of cooling and warming rates. Morphological survivals exceeded 80% when they were warmed at the highest rate (117,000°C/min) even when the prior cooling rate was as low as 880°C/min. Functional survival was >81% and 54% with the highest warming rate after cooling at 69,000 and 880°C/min, respectively. Our findings are also contrary to the second belief. We show that a high percentage of mouse oocytes survive vitrification in media that contain only half the usual concentration of solutes, provided they are warmed extremely rapidly; that is, >100,000°C/min. Again, the cooling rate is of less consequence.     

Chloroplast biogenesis — Correlation between structure and function

Chloroplast biogenesis is a multistage process leading to fully differentiated and functionally mature plastids. Complex analysis of chloroplast biogenesis was performed on the structural and functional level of its organization during the photoperiodic plant growth after initial growth of seedlings in the darkness. We correlated, at the same time intervals, the structure of etioplasts transforming into mature chloroplasts with the changes in the photosynthetic protein levels (selected core and antenna proteins of PSI and PSII) and with the function of the photosynthetic apparatus in two plant species: bean (Phaseolus vulgaris L.) and pea (Pisum sativum L). We selected these plant species since we demonstrated previously that the mature chloroplasts differ in the thylakoid organization. We showed that the protein biosynthesis as well as photosynthetic complexes formation proceeds gradually in both plants in spite of periods of darkness. We found that both steady structural differentiation of the bean chloroplast and reformation of prolamellar bodies in pea were accompanied by a gradual increase of the photochemical activity in both species. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Comparative effects of new generation oxazaphosphorines on the size and viability of human acute myeloblastic leukemia cells

Mafosfamide cyclohexylamine salt (D-17272), 4-hydro-peroxy-cyclophosphamide (D-18864), and beta-D-glucose-isophosphoramide mustard (D-19575, glufosfamide) are three new generation oxazaphosphorine agents. The aim of the present study was to compare the cell response to the action of these three oxazaphosphorines. The experiments were performed in vitro on human acute myeloblastic leukemia ML-1 cells. After exposure of ML-1 cells to the oxazaphosphorines, the size, viability and count of these cells were determined. The research was conducted using the spectrophotometric MTT assay and the electronic Beckman Coulter method. The temporary changes in the ML-1 cell size, viability and count, were dependent on the oxazaphosphorine agent tested, its dose, and the time intervals after its application. Among the three oxazaphosphorine agents, D-18864 proved to be the most cytotoxic, and D-19575 was characterized by the lowest cytotoxicity. The results suggest the possibility of using the electronic sizing and counting method and the MTT assay as a rapid in vitro test for assessing leukemic cell sensitivity to the action of new potential chemotherapeutic agents.     

Rhizobial plasmids — replication,structure and biological role

Soil bacteria, collectively named rhizobia, can establish mutualistic relationships with legume plants. Rhizobia often have multipartite genome architecture with a chromosome and several extrachromosomal replicons making these bacteria a perfect candidate for plasmid biology studies. Rhizobial plasmids are maintained in the cells using a tightly controlled and uniquely organized replication system. Completion of several rhizobial genome-sequencing projects has changed the view that their genomes are simply composed of the chromosome and cryptic plasmids. The genetic content of plasmids and the presence of some important (or even essential) genes contribute to the capability of environmental adaptation and competitiveness with other bacteria. On the other hand, their mosaic structure results in the plasticity of the genome and demonstrates a complex evolutionary history of plasmids. In this review, a genomic perspective was employed for discussion of several aspects regarding rhizobial plasmids comprising structure, replication, genetic content, and biological role. A special emphasis was placed on current post-genomic knowledge concerning plasmids, which has enriched the view of the entire bacterial genome organization by the discovery of plasmids with a potential chromosome-like role.     

Benzamide-based thiolcarbamates: a new class of HIV-1 NCp7 inhibitors

The HIV-1 nucleocapsid protein NCp7, which contains two highly conserved zinc fingers, is being used as a novel target for AIDS therapy due to its pivotal role in viral replication and its mutationally intolerant nature. Herein we report a new class of NCp7 inhibitors that possess good antiviral activity with low cellular toxicity.     

Ghrelin immunohistochemistry of gastric adenocarcinoma and mucoepidermoid carcinoma of salivary gland

Ghrelin (G-HH) synthesized in several tissues including salivary and stomach glands stimulates appetite in humans by modulating neuropeptide Y neurons in the hypothalamic arcuate nucleus. Loss of appetite is one of the most important symptoms of stomach cancer. We conducted a study using immunohistochemistry to determine whether salivary glands and stomach cancer tissues produce ghrelin. We determined that negative ghrelin immunohistochemistry discriminates tumors from normal tissues and may therefore further our understanding of the clinically important problem of reduced food intake and anorexia in cancer patients. Radioimmunoassay analyses confirmed that cancer cells do not produce a G-HH peptide, whereas normal cells yield this peptide.