A variety of changes,including CRISPR/Cas9‐mediated deletions,in CENH3 lead to haploid induction on outcrossing

Creating true‐breeding lines is a critical step in plant breeding. Novel, completely homozygous true‐breeding lines can be generated by doubled haploid technology in single generation. Haploid induction through modification of the centromere‐specific histone 3 variant (CENH3), including chimeric proteins, expression of non‐native CENH3 and single amino acid substitutions, has been shown to induce, on outcrossing to wild type, haploid progeny possessing only the genome of the wild‐type parent, in Arabidopsis thaliana. Here, we report the characterization of 31 additional EMS‐inducible amino acid substitutions in CENH3 for their ability to complement a knockout in the endogenous CENH3 gene and induce haploid progeny when pollinated by the wild type. We also tested the effect of double amino acid changes, which might be generated through a second round of EMS mutagenesis. Finally, we report on the effects of CRISPR/Cas9‐mediated in‐frame deletions in the αN helix of the CENH3 histone fold domain. Remarkably, we found that complete deletion of the αN helix, which is conserved throughout angiosperms, results in plants which exhibit normal growth and fertility while acting as excellent haploid inducers when pollinated by wild‐type pollen. Both of these technologies, CRISPR mutagenesis and EMS mutagenesis, represent non‐transgenic approaches to the generation of haploid inducers.     

Artificial evolution of OsEPSPS through an improved dual cytosine and adenine base editor generated a novel allele conferring rice glyphosate tolerance

Exploiting novel endogenous glyphosate-tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here,through fusions of different effective cytosine and adenine deaminases with nCas9-NG, we engineered an effective surrogate two-component composite base editing system, STCBE-2, with improved C-to-T and A-to-G base editing efficiency and expanded the editing window. Furthermore,we targeted a rice endogenous OsEPSPS gene for artificial evolution through ST...     

In vitro induction of early mouse embryo intracisternal particles (epsilon particles) in cultured cell lines.

The experimental induction of epsilon particles, retrovirus-like structures corresponding to the small IA particles of the mouse, was studied by electron microscopy in rodent-cultured cell lines. Among the chemicals tested, only IdUr was shown to be an effective inducer, but not cycloheximide, puromycin , deoxy-fluorouracil or 5-azacytidine. However, only two mouse-derived cell lines: Ki-BALB and FG 10, among 27 cell lines of mouse, rat and mink origins tested, expressed epsilon particles upon IdUr treatment. Epsilon particles thus respond to chemical inducers very differently in comparison with large IAP. Moreover, the addition of interferon previously shown to attenuate IAP production, had no effect on that of epsilon particles.     

Triiodothyronine (T3)-mediated toxicity and induction of apoptosis in insulin-producing INS-1 cells

Thyroid hormones reduce glucose tolerance in humans and animals. This effect is related to a decrease of glucose-induced insulin secretion following a reduction of pancreatic beta cell mass due to beta cell loss. The aim of this study was to analyze in vitro the mechanisms underlying the effects of triiodothyronine (T(3)) on the cell viability and cell cycle caused by changes of cell death or proliferation rate of insulin-producing INS-1 cells. 72-h Exposure of INS-1 cells to increasing T(3) concentrations up to 500 microM resulted in a significant viability reduction. This T(3) toxicity was caused by an increased apoptotic cell death rate, which was accompanied by a decreased proliferation rate. Inhibitory effects of T(3) on glucose-induced insulin secretion were already seen after 24 h of incubation, indicating that the deleterious effects of T(3) were time-dependent, changing from specific cellular dysfunctions to a severe and extended disturbance of the cellular survival program. Only T(3) concentrations higher than 250 microM were able to decrease cell viability and proliferation rate, to increase the rate of apoptosis and to reduce glucose-induced insulin secretion. These micromolar T(3) concentrations were significantly higher than the concentration range of T(3) receptor binding, indicating that other non-receptor-mediated mechanisms beyond the receptor level must be responsible for the observed toxic effects of T(3) in vitro.     

Photoperiodic regulation of receptacle induction in Sargassum horneri (Phaeophyceae) using clonal thalli

We developed a clonal culture of Sargassum horneri to investigate the effect of photoperiod on reproduction in this species. Regenerated vegetative thalli were obtained using lateral branches excised from a thallus grown from a single embryo under short‐day conditions (SD = 10:14 h light : dark cycle). Lateral branches excised from the SD‐regenerated thallus became vegetative thalli that remained in that phase as long as they were cultured under SD. When an excised lateral branch was cultured under long‐day conditions (LD = 14:10 h light : dark cycle), it began to enter the reproductive phase while still less than 50 mm long. Induction of the reproductive phase was accompanied by a distinctive morphological change – suppression of blade formation at the apical region of the branch; elongation of branches without blades was then followed by differentiation of receptacles bearing conceptacles on their surface. Apices of receptacles were able to interconvert between reproductive and vegetative phases, as blades resprouted upon transfer from LD to SD. The critical day length for induction of receptacle formation was between 13 and 14 h; receptacle formation was also induced under SD conditions with night breaks (NBs). These results strongly suggest that reproductive regulation of S. horneri is a photoperiodic long‐day response. NBs with blue and green light were effective for reproductive induction but not with red light. This suggests that blue‐ and/or green‐light photoreceptors are involved in the photoperiodic reproductive response of S. horneri.     

Spatial and temporal coincidence of induction processes and gap-junctional communication in Patella vulgata (Mollusca,Gastropoda)

In the early development of the gastropod mollusc Patella vulgata, two inductive processes take place between the animal and vegetal hemispheres. One is the induction of the vegetally-located stem cell of the mesoderm (the 3D macromere) by the first-quartet micromeres (which are located in the animal hemisphere). The other is the induction of dorsoventral symmetry in the first-quartet micromeres by the 3D macromere. As a consequence of the latter induction process, a dorsoventrally-organised prototroch is formed. In this paper, the moment of 3D induction is determined by deleting the inducing first-quartet micromeres at successive stages of development. Furthermore, the role of gap junctions in the two above-mentioned induction processes is investigated. This was done by studying lucifer yellow dye transfer between the 3D macromere and the first-quartet micromeres, in relation to these two induction processes. Analysis of the pattern of dye-transfer in vivo, in sections and with confocal laser scanning microscopy demonstrated that the moment of dye transfer coincides with the moment of 3D induction and with the moment of induction of the dorsoventral pattern of the prototroch. This indicates that gap-junctional communication may be involved in these two induction processes.     

TRAF6-mediated regulation of the PI3 kinase (PI3K)-Akt-GSK3beta cascade is required for TNF-induced cell survival

We recently demonstrated that the tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) helps maintenance of cell survival by regulating glycogen synthase kinase 3β (GSK3β) activity during TNF signaling. However, the molecular linkage between TRAF6 and GSK3β signaling is unknown. Herein, we showed that TRAF6 positively regulated cell survival by modulating PI3K-Akt-GSK3β cascades. In 3T3 cells lacking TRAF6, but not those lacking TRAF2, TNF stimulation led to prolonged hyperphosphorylation of Akt, which coincided with the activation of upstream PI3K. Pharmacologically blocking PI3K significantly inhibited Akt and GSK3β phosphorylation. Importantly, PI3K inhibition rescued cell death in TRAF6-null 3T3 cells. These data suggested TRAF6 regulates TNF-mediated cell survival through PI3K-Akt-GSK3β cascades.     

Acute in vivo treatment with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone does not alter base excision repair activities in murine lung and liver

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen found in unburned tobacco and tobacco smoke, and is believed to play an important role in human tobacco-induced cancers. In previous studies, NNK has been reported to induce oxidative DNA damage, and to alter DNA repair processes, effects that could contribute to pulmonary tumorigenesis in rodent models. The goal of this study was to determine the effects of NNK on levels of 8-hydroxydeoxyguanosine (8-OHdG), a biomarker of DNA oxidation, and activity of base excision repair (BER), which repairs oxidative DNA damage. Female A/J mice were treated with a tumorigenic dose of NNK (10 μmol) i.p. At 1, 2 and 24 h post treatment, there were no statistically significant differences in lung or liver 8-OHdG levels between control and NNK-treated mice (P > 0.05). Furthermore, NNK did not alter lung or liver BER activity compared to control at any time point (P > 0.05). In summary, acute treatment with a tumorigenic dose of NNK did not stimulate oxidative DNA damage or significantly alter BER activity, and these effects may not be major mechanisms of action of NNK in mouse models.     

Receptor for advanced glycation end products (RAGE)-mediated cytotoxicity of 3-hydroxypyridinium derivatives

Advanced glycation end products (AGEs) formed from glyceraldehyde (Gcer) and glycolaldehyde (Gcol) are involved in the pathogenesis of diabetic complications, via interactions with a receptor for AGEs (RAGE). In this study, we aimed to elucidate the RAGE-binding structure in Gcer and Gcol-derived AGEs and identify the minimal moiety recognized by RAGE. Among Gcer and Gcol-derived AGEs, GLAP (glyceraldehyde-derived pyridinium) and GA-pyridine elicited toxicity in PC12 neuronal cells. The toxic effects of GLAP and GA-pyridine were suppressed in the presence of anti-RAGE antibody or the soluble form of RAGE protein. Furthermore, the cytotoxicity test using GLAP analog compounds indicated that the 3-hydroxypyridinium (3-HP) structure is sufficient for RAGE-dependent toxicity. Surface plasmon resonance analysis showed that 3-HP derivatives directly interact with RAGE. These results indicate that GLAP and GA-pyridine are RAGE-binding epitopes, and that 3-HP, a common moiety of GLAP and GA-pyridine, is essential for the interaction with RAGE.     

Baculovirus expressed C-terminal fragment of bonnet monkey (Macaca radiata) zona pellucida glycoprotein-3 inhibits ZP3-mediated induction of acrosomal exocytosis

Zona pellucida glycoprotein-3 (ZP3) has been postulated as the primary sperm receptor in various mammalian species including bonnet monkey (Macaca radiata). However, information on the domain responsible for its binding to spermatozoa is inadequate. In the present study, bonnet monkey ZP3 (bmZP3), corresponding to amino acid (aa) residues 223-348 [bmZP3(223-348)] has been cloned and expressed using baculovirus expression system. SDS-PAGE and Western blot analysis of the purified renatured recombinant protein revealed it as a closely spaced doublet of approximately 25 kDa. Lectin-binding studies documented the presence of both O- as well as N-linked glycans. The biotinylated r-bmZP3(223-348) binds to the acrosomal region of the capacitated spermatozoa but fails to bind to the acrosome-reacted spermatozoa as investigated by immunofluorescence studies. In ELISA, nonbiotinylated r-bmZP3(223-348) and baculovirus expressed r-bmZP3, devoid of signal sequence and transmembrane-like domain [r-bmZP3(23-348)] competitively inhibit its binding to the capacitated spermatozoa. Interestingly, binding of biotinylated r-bmZP3(23-348) to the capacitated sperm is also inhibited by nonbiotinylated r-bmZP3(223-348). In contrast to r-bmZP3(23-348), r-bmZP3(223-348) failed to induce acrosomal exocytosis in the capacitated sperm. Interestingly, it competitively inhibits the acrosomal exocytosis induced by r-bmZP3(23-348). These studies, for the first time, identify a domain of ZP3 capable of binding to capacitated spermatozoa and inhibiting ZP3-mediated induction of acrosomal exocytosis furthering our understanding of mammalian fertilization.     

Excision Repair of 2,5-Diaziridinyl-1,4-Benzoquinone (DZQ)-DNA Adduct by Bacterial and Mammalian 3-Methyladenine-DNA Glycosylases

The mechanisms of anticancer activity of 2,5-diaziridinyl-1,4-benzoquinone (DZQ) are believed to involve the alkylation of guanine and adenine bases. In this study, it has been investigated whether bacterial and mammalian 3-methyladenine-DNA glycosylases are able to excise DZQ-DNA adduct with a differential substrate specificity. DZQ-induced DNA adduct was first formed in the radiolabeled restriction enzyme DNA fragment, and excision of the DNA adduct was analyzed following treatment with homogeneous 3-methyladenine-DNA glycosylase from E. coli, rat, and human, respectively. Abasic sites generated by DNA glycosylases were cleaved by the associated lyase activity of the E. coli formami-dopyrimidine-DNA glycosylase. Resolution of cleaved DNA on a sequencing gel with Maxam-Gilbert sequencing reactions showed that DZQ-induced adenine and guanine adducts were very good substrates for bacterial and mammalian enzymes. The E. coli enzyme excises DZQ-induced adenine and guanine adducts with similar efficiency. The rat and human enzymes, however, excise the adenine adduct more efficiently than the guanine adduct. These results suggest that the 3-methyladenine-DNA glycosylases from different origins have differential substrate specificity to release DZQ-DNA lesions. The use of 3-methyladenine-DNA glycosylase incision analysis could possibly be applied to quantify a variety of DNA adducts at the nucleotide level.     

The isolation and characterization of 3-(2-carboxyethyl)cytosine following in vitro reaction of β-propiolactone with calf thymus DNA

The new adduct 3-(2-carboxyethyl)cytosine (3-CEC) was isolated following in vitro reaction of the carcinogen β-propiolactone (BPL) with calf thymus DNA. The structure of 3-CEC was confirmed by synthesis from BPL and dCyd. Reaction of BPL with cCyd (pH 7.0–7.5, 37°C) gave 3-(2-carboxyethyl)deoxycytidine (3-CEdCyd) (9% yield) and 3,N⁴-bis(2-carboxyethyl)deoxycytidine (3,N⁴-BCEdCyd) (0.6% yield). 3-CEdCyd and 3,N⁴-BCEdCyd were hydrolyzed (1.5 N HC1, 100°C, 2 h) to 3-CEC and 3,N⁴-bis(2-carboxyethyl)cytosine (3,N⁴-BCEC), respectively. The structure of 3-CEC was assigned on the basis of UV and NMR spectra and the electron impact (EI) mass spectra of 3-CEC and a tri-trimethylsilyl (TMS) derivative of 3 CEC as well as deuterated (d₂₇) tri-TMS derivative of 3-CEC. The structure of 3,N⁴-BCEC was assigned on the basis of UV spectra and the EI mass spectra of a tri-TMS derivative. EI and isobutane chemical ionization mass spectra of 3-methylcytosine (3-MeCyt) and a di-TMS derivative of 3-MeCyt were obtained and were helpful in deducing the structures of 3-CEC and 3,N⁴-BCEC. This is the first report of the alkylation by BPL of an exocyclic atom on a base in DNA. Compound 3,N⁴-BCEC was not detected in BPL-reacted calf thymus DNA. The relative amounts of 1-(2-carboxyethyl)adenine (1-CEA), 7-(2-carboxyethyl)guanine (7-CEG), 3-(2-carboxyethyl)thymine (3-CET) and 3-CEC isolated from BPL-reacted DNA following perchloric acid hydrolysis were 0.23, 1.00, 0.39 and 0.41 respectively, when the alkylation reaction was conducted in phosphate buffer at 0–5°C and pH 7.5 and 0.10, 1.00, 0.29 and 0.28 respectively when the reaction was conducted in H₂O at 37°C and pH 7.0–7.5.     

Adventitious root formation in woody plant tissue: The influence of light and indole-3-butyric acid (IBA) on adventitious root induction in <Emphasis Type="Italic">Betula Pendula</Emphasis>

Summary The effects of auxin concentration and photoperiod on rooting were examined with a view to establishing a rooting regime for Betula pendula shoots cultured in vitro. Optimum concentrations of indole-3-butyric acid (IBA) were determined: the effects of a 16-h photoperiod and a pretreatment of 8d total darkness were examined. Maximum rooting rates and rooting densities (root number) were achieved using relatively low levels of IBA (0.39–0.74 μM). Both the dark and the light regimes produced roots, higher yields occurring with the latter. Maximum rooting percentage was reached after 30 d growth. in the light-treated cultures.     

Deletion of the 5-HT(3A)-receptor subunit blunts the induction of cocaine sensitization

Serotonin (5-HT) receptors are classified into seven groups (5-HT_1–7), comprising at least 14 structurally and pharmacologically distinct receptor subtypes. Pharmacological antagonism of ionotropic 5-HT₃ receptors has been shown to modulate both behavioral and neurochemical aspects of the induction of sensitization to cocaine. It is not known, however, if specific molecular subunits of the 5-HT₃ receptor influence the development of cocaine sensitization. To address this question, we studied the effects of acute and chronic intermittent cocaine administration in mice with a targeted deletion of the gene for the 5-HT_3A-receptor subunit (5-HT_3A−/−). 5-HT_3A (−/−) mice showed blunted induction of cocaine-induced locomotor sensitization as compared with wild-type littermate controls. 5-HT_3A (−/−) mice did not differ from wild-type littermate controls on measures of basal motor activity or response to acute cocaine treatment. Enhanced locomotor response to saline injection following cocaine sensitization was observed equally in 5-HT_3A (−/−) and wild-type mice suggesting similar conditioned effects associated with chronic cocaine treatment. These data show a role for the 5-HT_3A-receptor subunit in the induction of behavioral sensitization to cocaine and suggest that the 5-HT_3A molecular subunit modulates neurobehavioral adaptations to cocaine, which may underlie aspects of addiction.     

Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model

We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.