Sustained metaphase arrest in response to ionizing radiation in a non-small cell lung cancer cell line

Kodym, E., Kodym, R., Choy, H. and Saha, D. Sustained Metaphase Arrest in Response to Ionizing Radiation in a Non-small Cell Lung Cancer Cell Line. Radiat. Res. 169, 46-58 (2008). In solid tumors, non-apoptotic forms of tumor cell inactivation such as mitotic catastrophe appear to be predominant in the response to DNA-damaging agents. Despite its importance, the underlying molecular mechanisms of mitotic catastrophe have been only partially elucidated. We found that a large fraction of HCC2279 non-small cell lung cancer cells underwent mitotic catastrophe after irradiation. Cells were arrested in metaphase with chromosomal damage indicated by DNA fragments displaced from the metaphase plate and considerable numbers of residual gamma-H2AX foci. Although TP53 was nonfunctional, we detected a prompt radiation response on the level of checkpoint kinases. In contrast, CDC25A was the only checkpoint phosphatase that was responsive to radiation. CDC25B was not detectable, and CDC25C was constitutively phosphorylated at serine 216, leading to its cytoplasmic sequestration and functional inactivation. Therefore, radiation-induced mitotic catastrophe in HCC2279 cells appears to be induced by a combination of relative insufficiencies in the p53-mediated and checkpoint kinase-mediated pathways leading to premature entry into mitosis. Displaced chromosome fragments triggering an intra-M checkpoint in cells entering mitosis presumably result in a sustained metaphase arrest. The phenomenon found in these cells, which were derived directly from a human patient, might be responsible for therapy-induced genetic instability of tumors.     

Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: eukaryotic clones containing two and three ORF multi-gene cassettes expressed from a single promoter

Two types of eukaryotic operon-type Expression clones were constructed using the Multisite Gateway system employing six types of att signals. These clones harbored a DNA cassette containing two heterologous ORFs (cDNAs) or three heterologous ORFs in tandem downstream of a single promoter. The most promoter-proximal ORF was translated via a Kozak signal and the downstream one or two ORF(s) were translated as directed by internal ribosome entry site(s) (IRES). These clones were observed to produce two or three different proteins at levels that depended on the activities of the translational initiation signals used. With the intention of modulating the expression level of the first ORF, the translational initiation signals including a Kozak sequence and 11 different IRESs were investigated for their efficiency using a single ORF. The translational activity of these signals varied within a 10-fold magnitude. Using these results, expression at pre-described relative levels was achieved from the optional IRES of the respective ORFs in the cassette. Controllable expression at desired levels of two different ORFs directed by optional IRESs on a bicistronic construct, transcribed from a single promoter, was demonstrated.     

Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent

Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an alpha-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses alpha-smooth muscle actin, beta-actin, nonmuscle gamma-actin, and smooth muscle gamma-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during alpha-agonist contractions involves the remodeling of primarily gamma-actin and, to a lesser extent, beta-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.     

High salt‐treatment‐induced Na+ extrusion and low salt‐treatment‐induced Na+ accumulation in suspension‐cultured cells of the mangrove plant,Bruguiera sexangula

A suspension‐cultured cell strain of the mangrove plant (Bruguiera sexangula) was established from a callus culture and maintained in an amino acid medium in the absence of NaCl. NaCl non‐adapted cells were transferred to media containing 0–200 mm NaCl. The initial growth rate decreased gradually with increasing salt concentrations. However, at up to 150 mm NaCl, cell number growth at the highest point was almost the same as that at lower salt concentrations. Cells even continued to grow in the presence of 200 mm NaCl. Cells incubated in a medium containing 50 mm NaCl for 3 weeks accumulated Na⁺, while those incubated in 150 mm NaCl for 2 d showed only a transient increase in Na⁺ and Cl^– concentrations. In the latter treatment, the intracellular concentration of Na⁺ returned to the original low level within 2 weeks. It took a longer time for Cl^– to return to its original level. As a result, the Na⁺ and Cl^– concentrations in cells cultured with 50 mm NaCl were much larger than those in cells cultured with 150 mm NaCl. The intracellular distribution of ions after transfer to the medium containing 150 mm NaCl was analysed by isolating the vacuoles. Treatment with amiloride, an inhibitor of the Na⁺/H⁺ antiporter, suppressed the recovery of Na⁺ to the original level in the cells. Treatment with 150 mm NaCl for 3 d stimulated the activities of both the vanadate‐dependent H⁺‐ATPase and the Na⁺/H⁺ antiporter in the plasma membrane fraction.     

Improving salt tolerance of cotton seedlings with 5-aminolevulinic acid

Of 12 different plant growth regulators (PGRs) tested,5-aminolevulinic acid (ALA) was found to improve the salttolerance of cotton seedlings. Cotton seedlings treated with ALAcould grow in soil containing levels as high as 1.5% (wt/wt)NaCl. The analyses of mineral compositions of plant parts revealed that the Na^plus concentrations in the roots of the plantstreated with ALA were suppressed to low concentrations. Fromthese results, it can be presumed that the presence of ALA maycause a reduction of Na^plus uptake.     

Bimodal distribution of flowering time in a natural hybrid population of daylily (Hemerocallis fulva) and nightlily (Hemerocallis citrina)

Time of flower anthesis in a day is thought to evolve in response to the time of pollinator activities. We studied blooming and withering time in natural populations of daylily (Hemerocallis fulva), nightlily (Hemerocallis citrina) and their hybrids, and also in an artificially obtained array of the F1 hybrids. Blooming time of H. fulva varied from 4:30 to 7:30 and H. citrina varied from 16:30 to 20:30. In a natural hybrid population, blooming time and withering time showed discontinuous bimodal distribution in spite that morphological traits of flowers showed continuous unimodal variation. Most F1 hybrids showed diurnal flowering. These findings indicate that only a few genes have strong phenotypic effect on the determination of flowering time in Hemerocallis, and suggest that the evolution from a H. fulva-like ancestor to H. citrina was not a continuous process by accumulation of minute mutations.     

Full-length sequence analysis of subacute sclerosing panencephalitis (SSPE) virus, a mutant of measles virus, isolated from brain tissues of a patient shortly after onset of SSPE

Subacute sclerosing panencephalitis (SSPE) virus, a measles virus (MeV) mutant, was isolated from brain tissues of a patient shortly after the clinical onset, and the entire viral genome was sequenced. The virus, named SSPE-Kobe-1, formed syncytia on B95a and Vero/SLAM cells without producing cell-free infectious virus particles, which is characteristic of SSPE virus. Phylogenetic analysis classified SSPE-Kobe-1 into genotype D3. When compared with an MeV field isolate of the same genotype (Ich-B strain), SSPE-Kobe-1 exhibited mutation rates of 0.8-1.6% at the nucleotide level in each of the proteincoding regions of the viral genome. It is noteworthy that the mutation rate of the M gene (1.2%) of SSPE-Kobe-1 was considerably lower than for other SSPE virus strains reported so far, but that the majority of the mutations (75%) were the uridine-to-cytidine biased hypermutation characteristic of the SSPE virus M gene. At the amino acid level, the viral proteins, such as N, P, C, V, M, F, H and L proteins, had point-mutations on 3, 7, 1, 4, 3, 9, 8 and 14 residues, respectively, compared with the Ich-B strain. In addition, the F and H proteins had mutated C-termini due to single-point mutations near or at the stop codons. Two of the three mutations in the M protein were Leu-to-Pro mutations, which are likely to affect the conformation and, therefore, the function of the protein. Because of the relatively small number of mutations, SSPE-Kobe-1 would be a useful tool to study genetic evolution of SSPE virus.     

Protein kinase B inhibits endostatin-induced apoptosis in HUVECs

Endostatin is a tumor-derived angiogenesis inhibitor, and the endogenous 20 kDa carboxyl-terminal fragment of collagen XVIII. In addition to inhibiting angiogenesis,endostatin inhibits tumor growth and the induction of apoptosis in several endothelial cell types. However, the mechanisms that regulate endostatin-induced apoptotic cell death are unclear. Here, we investigated apoptotic cell death and the underlying regulatory mechanisms elicited of endostatin in human umbilical vein endothelial cells (HUVECs). Endostatin was found to induce typical apoptotic features, such as, chromatin condensation and DNA fragmentation in these cells. Thus, as the phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in various cell types, we investigated whether this pathway could protect cells against endostatin induced apoptosis. It was found that the inhibition of PI3K/PKB significantly increased endostatin-induced apoptosis, and that endostatininduced cell death is physiologically linked to PKB-mediated cell survival through caspase-8.     

Biochemical and immunohistochemical analyses of GnRH-like peptides in the nerve ganglion of the chiton, Acanthopleura japonica

We examined whether a gonadotropin-releasing hormone (GnRH)-like peptide is present in the nerve ganglion of the chiton Acanthopleura japonica (Mollusca, Polyplacophora) using reverse-phase high performance liquid chromatography (rpHPLC) combined with time-resolved fluoroimmunoas-say (TR-FIA) analysis, and immunohistochemistry. An extract of the chiton head region showed a similar retention time to that of synthetic lamprey GnRH-II on rpHPLC combined with TR-FIA analysis using a rabbit polyclonal antibody raised against chicken GnRH-II (aCII6). Cell bodies immunostained with LRH13 (a mouse monoclonal antibody raised against the common amino acid sequence of vertebrate GnRH) were detected in the cerebrobuccal ring (CBR). Cell bodies immunostained with aCII6 were not only observed in the CBR but also in the lateral nerve cord (LCo). Fibers immunostained with LRH13 and aCII6 were widely distributed throughout the central nervous system in the CBR, subradular ganglion (SubRG), pedal nerve cord (PCo), pedal commissure (P/PCom), lateropedal commissure (L/PCom), and from the LCo to the suprarectal commissure (SupRecCom). The cell bodies and fibers immunostained with these two antisera were distinguishable by dual-label immunohistochemistry. These results suggest that multiple GnRH-like peptides are present in the nerve ganglion of the chiton Acanthopleura japonica.     

Development of 3-D poly(trimethylenecarbonate-co-ε-caprolactone)-block-poly(p-dioxanone) scaffold for bone regeneration with high porosity using a wet electrospinning method

The three dimensional (3-D) poly(trimethylenecarbonate-co-ε-caprolactone)-block-poly(p-dioxanone) scaffold was made using a wet electrospinning method and its application as a tissue engineered matrix was evaluated for bone regeneration. The scaffold was highly porous (90%) and interconnected among pores. Under scanning electron microscopy, the cells of the center of the scaffold showed healthy well attached shape even at 4 days after seeding. The osteoblastic MC3T3-E1 cells proliferated 1.2 times faster at 4 day, 1.5 times faster at 7 days after seeding as compared with the control in the scaffold (P < 0.05). The activity of alkaline phosphatase, a bone formation marker, of cells seeded in the scaffold was nearly four times faster compared to control 28 days after seeding (P < 0.05). Taken together, newly developed 3-D poly(trimethylenecarbonate-co-ε-caprolactone)-block-poly(p-dioxanone) scaffold is a promising candidate for bone regeneration.