Carcinogens can induce homologous recombination between duplicated chromosomal sequences in mouse L cells.

The ability of a series of DNA-damaging agents to induce homologous intrachromosomal recombination between duplicated genes in the chromosome of mouse cells was investigated. The target cells were the thymidine kinase-deficient mouse L-cell strain 333M, which contains a single integrated copy of a plasmid with two herpes simplex virus thymidine kinase (Htk) genes, each containing an 8-base-pair XhoI linker inserted at a unique site. Expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. The spontaneous rate of recombination in this strain is 3 per 10(6) cells per generation. The agents tested represent physical carcinogens (UV and ionizing radiation), a simple alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), an alkylating cross-linking agent (mitomycin C), and a reactive metabolite of a polycyclic aromatic hydrocarbon ((+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [BPDE] ). The background frequency of tk+ recombinants in the untreated population averaged 18 X 10(-6) +/- 5 X 10(-6). Ionizing radiation had little or no effect on recombination; exposure to mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, BPDE, or UV, at doses that lowered the survival to between 90 and 10% of the control, caused a dose-dependent increase in frequency of recombinants, reaching 50 X 10(-6) to 100 X 10(-6). No tk+ cells could be generated with a control cell line that contained only one mutant copy of the Htk gene. Molecular hybridization analysis showed that 85 to 90% of the tk+ recombinants retained the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information, gene conversion. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. Each recombinant tested contained an XhoI-resistant (wild-type) Htk gene.     

Cloning and characterization of a SnRK1-encoding gene from <Emphasis Type="Italic">Malus hupehensis</Emphasis> Rehd. and heterologous expression in tomato

Sucrose non-fermenting-1-related protein kinase-1 (SnRK1) plays an important role in metabolic regulation in plant. To understand the molecular mechanism of amino acids and carbohydrate metabolism in Malus hupehensis Rehd. var. pinyiensis Jiang (Pingyi Tiancha, PYTC), a full-length cDNA clone encoding homologue of SnRK1 was isolated from PYTC by Rapid Amplification of cDNA Ends (RACE). The clone, designated as MhSnRK1, contains 2063 nucleotides with an open reading frame of 1548 nucleotides. The deduced 515 amino acids showed high identities with other plant SnRK1 genes. Quantitative real-time PCR analysis revealed this gene was expressed in roots, stems and leaves. Exposing seedlings to nitrate caused and initial decrease in expression of the MhSnRK1 gene in roots, leaves and stems in short term. Ectopic expression of MhSnRK1 in tomato mainly resulted in higher starch content in leaf and red-ripening fruit than wild-type plants. This result supports the hypothesis that overexpression of SnRK1 causes the accumulation of starch in plant cells. All the results suggest that MhSnRK1 may play important roles in carbohydrate and amino acid metabolisms.     

Intra- and inter-specific effects of mast seeding on seed fates of two sympatric <Emphasis Type="Italic">Corylus</Emphasis> species

We released seeds of two sympatric tree species, Corylus mandshurica (seed with thinner seed hull, higher nutrition) and C. heterophylla (seeds with thicker seed hull, lower nutrition) in the masting year of C. mandshurica in 2008, and C. heterophylla in 2009, respectively, to investigate how seed masting of the two sympatric Corylus species affects seed removal and dispersal fitness of the two species differently at both intra- and inter-specific levels. At intra-specific level, the authors found mast seeding of both C. mandshurica and C. heterophylla significantly reduced seed removal, seed consumption, but increased seed dispersal distance and seed dispersal fitness of the released seeds. Mast seeding of C. mandshurica increased seed caching of C. mandshurica. At inter-specific level, the authors found mast seeding of C. mandshurica reduced seed removal of C. heterophylla, but mast seeding of C. heterophylla did not significantly reduce seed removal of C. mandshurica. Mast seeding of C. mandshurica reduced seed consumption of C. heterophylla, while mast seeding of C. heterophylla reduced seed consumption of C. mandshurica. We found mast seeding of C. mandshurica significantly reduced seed dispersal distance of C. heterophylla, while mast seeding of C. heterophylla significantly increased seed dispersal distance of C. mandshurica. We found that mast seeding of C. mandshurica significantly increased seed dispersal fitness of C. heterophylla, while mast seeding of C. heterophylla did not significantly increase seed dispersal fitness of C. mandshurica. More studies are needed to reveal the ecological consequences of mast seeding at inter-specific or community-level. Seed traits may attribute the differences of mast seeding at inter-specific level. Because seeds with thinner seed hull and higher nutrition were more harvested and eaten by rodents, mast seeding of C. mandshurica might have reduced seed removal and seed consumption, but increased dispersal fitness of C. heterophylla (seeds with thicker seed hull, lower nutrition). Therefore, synchrony among species is, or is not, selectively beneficial to the focus species depends on seed traits which determine gains from mast seeding at inter-specific level.     

Thermodynamic and sequential characteristics of phase separation and droplet formation for an intrinsically disordered region/protein ensemble

Liquid–liquid phase separation (LLPS) of some IDPs/IDRs can lead to the formation of the membraneless organelles in vitro and in vivo, which are essential for many biological processes in the cell. Here we select three different IDR segments of chaperon Swc5 and develop a polymeric slab model at the residue-level. By performing the molecular dynamics simulations, LLPS can be observed at low temperatures even without charge interactions and disappear at high temperatures. Both the sequence length and the charge pattern of the Swc5 segments can influence the critical temperature of LLPS. The results suggest that the effects of the electrostatic interactions on the LLPS behaviors can change significantly with the ratios and distributions of the charged residues, especially the sequence charge decoration (SCD) values. In addition, three different forms of swc conformation can be distinguished on the phase diagram, which is different from the conventional behavior of the free IDP/IDR. Both the packed form (the condensed-phase) and the dispersed form (the dilute-phase) of swc chains are found to be coexisted when LLPS occurs. They change to the fully-spread form at high temperatures. These findings will be helpful for the investigation of the IDP/IDR ensemble behaviors as well as the fundamental mechanism of the LLPS process in bio-systems.     

Sphingosine kinase 1 regulates HMGB1 translocation by directly interacting with calcium/calmodulin protein kinase II-δ in sepsis-associated liver injury

Previously, we confirmed that sphingosine kinase 1 (SphK1) inhibition improves sepsis-associated liver injury. High-mobility group box 1 (HMGB1) translocation participates in the development of acute liver failure. However, little information is available on the association between SphK1 and HMGB1 translocation during sepsis-associated liver injury. In the present study, we aimed to explore the effect of SphK1 inhibition on HMGB1 translocation and the underlying mechanism during sepsis-associated liver injury. Primary Kupffer cells and hepatocytes were isolated from SD rats. The rat model of sepsis-associated liver damage was induced by intraperitoneal injection with lipopolysaccharide (LPS). We confirmed that Kupffer cells were the cells primarily secreting HMGB1 in the liver after LPS stimulation. LPS-mediated HMGB1 expression, intracellular translocation, and acetylation were dramatically decreased by SphK1 inhibition. Nuclear histone deacetyltransferase 4 (HDAC4) translocation and E1A-associated protein p300 (p300) expression regulating the acetylation of HMGB1 were also suppressed by SphK1 inhibition. HDAC4 intracellular translocation has been reported to be controlled by the phosphorylation of HDAC4. The phosphorylation of HDAC4 is modulated by CaMKII-δ. However, these changes were completely blocked by SphK1 inhibition. Additionally, by performing coimmunoprecipitation and pull-down assays, we revealed that SphK1 can directly interact with CaMKII-δ. The colocalization of SphK1 and CaMKII-δ was verified in human liver tissues with sepsis-associated liver injury. In conclusion, SphK1 inhibition diminishes HMGB1 intracellular translocation in sepsis-associated liver injury. The mechanism is associated with the direct interaction of SphK1 and CaMKII-δ.Subject terms: Hepatotoxicity, Sepsis     

Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells.

Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell.     

Regulation of beta-endorphin and ACTH in brain by estradiol

The effect of estradiol on the brain concentration of immunoreactive beta-endorphin (beta-EP) and C-terminal ACTH (CLIP) was studied in ovariectomized rats. Dopamine, a known inhibitor of pituitary intermediate lobe pro-opiomelanocortin (POMC), was examined as a possible mediator of the estradiol induced changes in brain POMC. Animals were treated for 1 or 3 weeks with either 1) saline; 2) silastic estradiol implants; or 3) estradiol implants plus haloperidol 1 mg/kg/day. After one week of treatment no significant change in hypothalamic beta-EP content was noted in any group compared to the control level of 4.13 +/- .33 (SEM) pmoles although in the neurointermediate lobe beta-EP increased from 566 +/- 72 to 942 +/- 73 pmoles after haloperidol (p less than .005). After 3 weeks, however, hypothalamic beta-EP decreased from 3.96 +/- .28 to 2.74 +/- .19 pmoles (p less than .005) and C-terminal ACTH decreased from 3.78 +/- .33 to 2.82 +/- .18 pmoles (p less than .02) in the estradiol treated rats. This estradiol induced decrease in the hypothalamic content of beta-EP and C-terminal ACTH was not blocked by haloperidol. We conclude that estradiol lowers the hypothalamic content of beta-EP and CLIP and that this effect does not appear to be mediated by dopamine.     

Microplastics in soil-plant system: effects of nano/microplastics on plant photosynthesis,rhizosphere microbes and soil properties in soil with different residues

Plant and Soil - To investigate the effects of polystyrene microplastics (PS-beads) on the soil properties, photosynthesis of Flowering Chinese cabbage, the rhizosphere microbial community and...     

Single-cell landscape of the ecosystem in early-relapse hepatocellular carcinoma

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