SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

Background  

Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study.     

High susceptibility of neural stem cells to methylmercury toxicity: effects on cell survival and neuronal differentiation

Neural stem cells (NSCs) play an essential role in both the developing embryonic nervous system through to adulthood where the capacity for self-renewal may be important for normal function of the CNS, such as in learning, memory and response to injury. There has been much excitement about the possibility of transplantation of NSCs to replace damaged or lost neurones, or by recruitment of endogenous precursors. However, before the full potential of NSCs can be realized, it is essential to understand the physiological pathways that control their proliferation and differentiation, as well as the influence of extrinsic factors on these processes. In the present study we used the NSC line C17.2 and primary embryonic cortical NSCs (cNSCs) to investigate the effects of the environmental contaminant methylmercury (MeHg) on survival and differentiation of NSCs. The results show that NSCs, in particular cNSCs, are highly sensitive to MeHg. MeHg induced apoptosis in both models via Bax activation, cytochrome c translocation, and caspase and calpain activation. Remarkably, exposure to MeHg at concentrations comparable to the current developmental exposure (via cord blood) of the general population in many countries inhibited spontaneous neuronal differentiation of NSCs. Our studies also identified the intracellular pathway leading to MeHg-induced apoptosis, and indicate that NSCs are more sensitive than differentiated neurones or glia to MeHg-induced cytotoxicity. The observed effects of MeHg on NSC differentiation offer new perspectives for evaluating the biological significance of MeHg exposure at low levels.     

Genetic Perturbation of the Maize Methylome

DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward- or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context- and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana.     

Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): patterns of variation consistent with reticulate evolution

The ITS sequences of Acropora spp. are the shortest so far identified in any metazoan and are among the shortest seen in eukaryotes; ITS1 was 70-80 bases, and ITS2 was 100-112 bases. The ITS sequences were also highly variable, but base composition and secondary structure prediction indicate that divergent sequence variants are unlikely to be pseudogenes. The pattern of variation was unusual in several other respects: (1) two distinct ITS2 types were detected in both A. hyacinthus and A. cytherea, species known to hybridize in vitro with high success rates, and a putative intermediate ITS2 form was also detected in A. cytherea; (2) A. valida was found to contain highly (29%) diverged ITS1 variants; and (3) A. longicyathus contained two distinct 5.8S rDNA types. These data are consistent with a reticulate evolutionary history for the genus Acropora.      

Getting the right stuff： controlling neural stem cell state and fate in vivo and in vitro with biomaterials

Stem cell therapy holds great promises in medical treatment by, e.g., replacing lost cells, re-constitute healthy cell populations and also in the use of stem cells as vehicles for factor and gene delivery. Embryonic stem cells have rightfully attracted a large interest due to their proven capacity of differentiating into any cell type in the embryo in vivo. Tissue-specific stem ceils are however already in use in medical practice, and recently the first systematic medical trials involving human neural stem cell （NSC） therapy have been launched. There are yet many obstacles to overcome and procedures to improve. To ensure progress in the medical use of stem cells increased basic knowledge of the molecular mechanisms that govern stem cell characteristics is necessary. Here we provide a review of the literature on NSCs in various aspects of cell therapy, with the main focus on the potential of using biomaterials to control NSC characteristics, differentiation, and delivery. We summarize results from studies on the characteristics of endogenous and transplanted NSCs in rodent models of neurological and cancer diseases, and highlight recent advancements in polymer compatibility and applicability in regulating NSC state and fate. We suggest that the development of specially designed polymers, such as hydrogels, is a crucial issue to improve the outcome of stem cell therapy in the central nervous system.     

Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [γ-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [γ-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.     

Glucocorticoids induce long-lasting effects in neural stem cells resulting in senescence-related alterations

Alterations in intrauterine programming occurring during critical periods of development have adverse consequences for whole-organ systems or individual tissue functions in later life. In this paper, we show that rat embryonic neural stem cells (NSCs) exposed to the synthetic glucocorticoid dexamethasone (Dex) undergo heritable alterations, possibly through epigenetic mechanisms. Exposure to Dex results in decreased NSC proliferation, with no effects on survival or differentiation, and changes in the expression of genes associated with cellular senescence and mitochondrial functions. Dex upregulates cell cycle-related genes p16 and p21 in a glucocorticoid receptor(GR)-dependent manner. The senescence-associated markers high mobility group (Hmg) A1 and heterochromatin protein 1 (HP1) are also upregulated in Dex-exposed NSCs, whereas Bmi1 (polycomb ring finger oncogene) and mitochondrial genes Nd3 (NADH dehydrogenase 3) and Cytb (cytochrome b) are downregulated. The concomitant decrease in global DNA methylation and DNA methyltransferases (Dnmts) suggests the occurrence of epigenetic changes. All these features are retained in daughter NSCs (never directly exposed to Dex) and are associated with a higher susceptibility to oxidative stress, as shown by the increased occurrence of apoptotic cell death on exposure to the redox-cycling reactive oxygen species (ROS) generator 2,3-dimethoxy-1-naphthoquinone (DMNQ). Our study provides novel evidence for programming effects induced by glucocorticoids (GCs) on NSCs and supports the idea that fetal exposure to endogenous or exogenous GCs is likely to result in long-term consequences that may predispose to neurodevelopmental and/or neurodegenerative disorders.     

Correlation of myosin isoforms with anatomical divisions in equine musculus biceps brachii.

The biceps brachii of horses is subdivided into a lateral and medial head. Electrophoresis of samples from the lateral head revealed three slow-migrating native myosin isoforms, including one that does not correspond to slow myosin isoforms described for other mammalian muscles. In contrast, the medial head contained a single slow isoform. Both the lateral and medial heads contained three fast-migrating isoforms corresponding with the FM-2, FM-3 and FM-4 isoforms reported for other mammalian fast-twitch muscle fibers. Electrophoresis of myosin heavy chains (MHCs) revealed only two MHC bands, one fast-migrating band that comigrates with rat type I MHC and a second slower-migrating band that comigrates with rat type IIa MHC. Quantitation of the histochemical data is correlated with densitometric analysis of MHCs in the medial and lateral heads of biceps brachii and is consistent with previously hypothesized functional specializations of this muscle.     

Continuous-time modeling of cell fate determination in Arabidopsis flowers

Background  

The genetic control of floral organ specification is currently being investigated by various approaches, both experimentally and through modeling. Models and simulations have mostly involved boolean or related methods, and so far a quantitative, continuous-time approach has not been explored.     

Strategy for Identification of Novel Fungal and Bacterial Glycosyl Hydrolase Hybrid Mixtures that can Efficiently Saccharify Pretreated Lignocellulosic Biomass

A rational four-step strategy to identify novel bacterial glycosyl hydrolases (GH), in combination with various fungal enzymes, was applied in order to develop tailored enzyme cocktails to efficiently hydrolyze pretreated lignocellulosic biomass. The fungal cellulases include cellobiohydrolase I (CBH I; GH family 7A), cellobiohydrolase II (CBH II; GH family 6A), endoglucanase I (EG I; GH family 7B), and β-glucosidase (βG; GH family 3). Bacterial endocellulases (LC1 and LC2; GH family 5), β-glucosidase (LβG; GH family 1), endoxylanases (LX1 and LX2; GH family 10), and β-xylosidase (LβX; GH family 52) from multiple sources were cloned, expressed, and purified. Enzymatic hydrolysis for varying enzyme combinations was carried out on ammonia fiber expansion (AFEX)-treated corn stover at three total protein loadings (i.e., 33, 16.5, and 11 mg enzyme/g glucan). The optimal mass ratio of enzymes necessary to maximize both glucan and xylan yields was determined using a suitable design of experiments. The optimal hybrid enzyme mixtures contained fungal cellulases (78% of total protein loading), which included CBH I (loading ranging between 9-51% of total enzyme), CBH II (9-51%), EG I (10-50%), and bacterial hemicellulases (22% of total protein loading) comprising of LX1 (13%) and LβX (9%). The hybrid mixture was effective at 50°C, pH 4.5 to maximize saccharification of AFEX-treated corn stover resulting in 95% glucan and 65% xylan conversion. This strategy of screening novel enzyme mixtures on pretreated lignocellulose would ultimately lead to the development of tailored enzyme cocktails that can hydrolyze plant cell walls efficiently and economically to produce cellulosic ethanol.