Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA)

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe context-dependent assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA-generated ZFNs, we rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multigene pathways or genome-wide alterations.     

Role of cyclin B1/Cdc2 up-regulation in the development of mitotic prometaphase arrest in human breast cancer cells treated with nocodazole

Background

During a normal cell cycle, the transition from G₂ phase to mitotic phase is triggered by the activation of the cyclin B1-dependent Cdc2 kinase. Here we report our finding that treatment of MCF-7 human breast cancer cells with nocodazole, a prototypic microtubule inhibitor, results in strong up-regulation of cyclin B1 and Cdc2 levels, and their increases are required for the development of mitotic prometaphase arrest and characteristic phenotypes.

Methodology/Principal Findings

It was observed that there was a time-dependent early increase in cyclin B1 and Cdc2 protein levels (peaking between 12 and 24 h post treatment), and their levels started to decline after the initial increase. This early up-regulation of cyclin B1 and Cdc2 closely matched in timing the nocodazole-induced mitotic prometaphase arrest. Selective knockdown of cyclin B1or Cdc2 each abrogated nocodazole-induced accumulation of prometaphase cells. The nocodazole-induced prometaphase arrest was also abrogated by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or with cycloheximide, a protein synthesis inhibitor that was found to suppress cyclin B1 and Cdc2 up-regulation. In addition, we found that MAD2 knockdown abrogated nocodazole-induced accumulation of cyclin B1 and Cdc2 proteins, which was accompanied by an attenuation of nocodazole-induced prometaphase arrest.

Conclusions/Significance

These observations demonstrate that the strong early up-regulation of cyclin B1 and Cdc2 contributes critically to the rapid and selective accumulation of prometaphase-arrested cells, a phenomenon associated with exposure to microtubule inhibitors.     

New diterpene furanoids from the Antarctic lichen Huea sp

In the course of ongoing research on protein tyrosine phosphatase 1B (PTP1B) inhibitory compounds from Antarctic lichens, four new diterpene furanoids, hueafuranoids A–D (1–4) have been isolated from the MeOH extract of Antarctic lichen Huea sp. by various chromatographic methods. The structures of these compounds were elucidated by analysis of NMR and MS data, and comparing their spectral data with those in the literature. Compound 1 showed inhibitory activity against therapeutically targeted protein, PTP1B with an IC₅₀ value of 13.9 μM. The kinetic analysis of PTP1B inhibition by hueafuranoid A (1) suggested that the diterpene furanoids encountered in this study inhibited PTP1B activity in a non-competitive manner.     

Polar and Alpine Microbial Collection (PAMC): a culture collection dedicated to polar and alpine microorganisms

Microorganisms in polar areas may have important ecological roles in biogeochemical cycles and the food chain. They are adapted to polar environments by means of special physiological adaptation mechanisms that include cold-adapted enzymes and cryoprotectants such as exopolysaccharides. Culture collections for polar microorganisms can provide research resources for ecological and physiological studies. The Polar and Alpine Microbial Collection (PAMC) is a specialized culture collection for maintenance and distribution of polar and alpine microorganisms. A database system was developed to share important data fields with DarwinCore2 and Ocean Biogeographic Information System database schemas. Approximately 1,500 out of 5,500 strains maintained in PAMC have been identified and belonged primarily to the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Many of the microbial strains can grow at low temperature and produce proteases, lipases, and/or exopolysaccharides. PAMC provides search tools based on keywords such as taxonomy, geographical origin, habitat, and physiological characteristics. Biological materials and information provided by PAMC will be important resources for ecological and physiological studies on polar and alpine microorganisms.     

Optimal investment for enhancing social concern about biodiversity conservation: A dynamic approach

To maintain biodiversity conservation areas, we need to invest in activities, such as monitoring the condition of the ecosystem, preventing illegal exploitation, and removing harmful alien species. These require a constant supply of resources, the level of which is determined by the concern of the society about biodiversity conservation. In this paper, we study the optimal fraction of the resources to invest in activities for enhancing the social concern y(t) by environmental education, museum displays, publications, and media exposure. We search for the strategy that maximizes the time-integral of the quality of the conservation area x(t) with temporal discounting. Analyses based on dynamic programming and Pontryagin’s maximum principle show that the optimal control consists of two phases: (1) in the first phase, the social concern level approaches to the final optimal value y^∗, (2) in the second phase, resources are allocated to both activities, and the social concern level is kept constant y(t)=y^∗. If the social concern starts from a low initial level, the optimal path includes a period in which the quality of the conservation area declines temporarily, because all the resources are invested to enhance the social concern. When the support rate increases with the quality of the conservation area itself x(t) as well as with the level of social concern y(t), both variables may increase simultaneously in the second phase. We discuss the implication of the results to good management of biodiversity conservation areas.     

PDIp is a major intracellular oestrogen-storage protein that modulates tissue levels of oestrogen in the pancreas

E2 (17β-oestradiol), a female sex hormone, has important biological functions in a woman's body. The pancreas, often considered a non-classical E2-targeting organ, is known to be functionally regulated by E2, but little is known about how oestrogen actions are regulated in this organ. In the present study we report that PDIp (pancreas-specific protein disulfide isomerase), a protein-folding catalyst, can act as a major intracellular E2 storage protein in a rat model to modulate the pancreatic tissue level, metabolism and action of E2. The purified endogenous PDIp from both rat and human pancreatic tissues can bind E2 with a Kd value of approximately 150?nM. The endogenous PDIp-bound E2 accounts for over 80% of the total protein-bound E2 present in rat and human pancreatic tissues, and this binding protects E2 from metabolic disposition and prolongs its duration of action. Importantly, we showed in ovariectomized female rats that the E2 level in the pancreas reaches its highest level (9-fold increase over its basal level) at 24-48?h after a single injection of E2, and even at 96?h its level is still approximately 5-fold higher. In contrast, the E2 level in the uterus quickly returns to its basal level at 48?h after reaching its maximal level (approximately 2-fold increase) at 24?h. Taken together, these results show for the first time that PDIp is a predominant intracellular oestrogen storage protein in the pancreas, which offers novel mechanistic insights into the accumulation and action of oestrogen inside pancreatic cells.     

CaMKII inhibition in heart failure, beneficial, harmful, or both

Calmodulin-dependent protein kinase II (CaMKII) has been proposed to be a therapeutic target for heart failure (HF). However, the cardiac effect of chronic CaMKII inhibition in HF has not been well understood. We have tested alterations of Ca(2+) handling, excitation-contraction coupling, and in vivo β-adrenergic regulation in pressure-overload HF mice with CaMKIIδ knockout (KO). HF was produced in wild-type (WT) and KO mice 1 wk after severe thoracic aortic banding (sTAB) with a continuous left ventricle (LV) dilation and reduction of ejection fraction for up to 3 wk postbanding. Cardiac hypertrophy was similar between WT HF and KO HF mice. However, KO HF mice manifested exacerbation of diastolic function and reduction in cardiac reserve to β-adrenergic stimulation. Compared with WT HF, L-type calcium channel current (I(Ca)) density in KO HF LV was decreased without changes in I(Ca) activation and inactivation kinetics, whereas I(Ca) recovery from inactivation was accelerated and Ca(2+)-dependent I(Ca) facilitation, a positive staircase blunted in WT HF, was recovered. However, I(Ca) response to isoproterenol was reduced. KO HF myocytes manifested dramatic decrease in sarcoplasmic reticulum (SR) Ca(2+) leak and slowed cytostolic Ca(2+) concentration decline. Sarcomere shortening was increased, but relaxation was slowed. In addition, an increase in myofilament sensitivity to Ca(2+) and the slow skeletal muscle troponin I-to-cardiac troponin I ratio and interstitial fibrosis and a decrease in Na/Ca exchange function and myocyte apoptosis were observed in KO HF LV. CaMKIIδ KO cannot suppress severe pressure-overload-induced HF. Although cellular contractility is improved, it reduces in vivo cardiac reserve to β-adrenergic regulation and deteriorates diastolic function. Our findings challenge the strategy of CaMKII inhibition in HF.     

Lysine 263 residue of NPM/B23 is essential for regulating ATP binding and B23 stability

Here, we show that Nucleophsomin/B23 provides lysine 263 as a critical binding site for ATP. Mutagenesis of lysine 263 to asparagine (K263N) disrupts B23 from ATP binding. While B23 WT exclusively localizes to the nucleolus, the B23-K263N is redistributed from the nucleolus to the nucleoplam. Notably, the K263N mutant is unstable, and displayed rapid degradation. Alteration of K263 induced B23 instability through increased ubiquitination and proteaosomal degradation. Moreover, mutation of K263 impedes the mitogenic effect of B23 in PC12 cells. Thus, K263 is a critical site for ATP binding and required for B23 stability, confining B23 in the nucleolus.