Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects

Engineered zinc finger nucleases (ZFNs) induce DNA double-strand breaks at specific recognition sequences and can promote efficient introduction of desired insertions, deletions or substitutions at or near the cut site via homology-directed repair (HDR) with a double- and/or single-stranded donor DNA template. However, mutagenic events caused by error-prone non-homologous end-joining (NHEJ)-mediated repair are introduced with equal or higher frequency at the nuclease cleavage site. Furthermore, unintended mutations can also result from NHEJ-mediated repair of off-target nuclease cleavage sites. Here, we describe a simple and general method for converting engineered ZFNs into zinc finger nickases (ZFNickases) by inactivating the catalytic activity of one monomer in a ZFN dimer. ZFNickases show robust strand-specific nicking activity in vitro. In addition, we demonstrate that ZFNickases can stimulate HDR at their nicking site in human cells, albeit at a lower frequency than by the ZFNs from which they were derived. Finally, we find that ZFNickases appear to induce greatly reduced levels of mutagenic NHEJ at their target nicking site. ZFNickases thus provide a promising means for inducing HDR-mediated gene modifications while reducing unwanted mutagenesis caused by error-prone NHEJ.     

Purification and identification of an angiotensin I-converting enzyme inhibitory peptide from fermented soybean extract

Angiotensin I-converting enzyme (ACE), a dipeptidyl carboxypeptidase, plays an important physiological role in regulating blood pressure. ACE-inhibitory peptides derived from food proteins have potential pharmaceutical and human health uses. In this study, we prepared a fermented soybean extract (FSE) through a rapid fermentation at an elevated temperature to accelerate proteolytic hydrolysis and described purification procedures to discover potent ACE-inhibitory peptides from FSE. After 3 days of aging, FSE exhibited ACE-inhibitory activity with an IC₅₀ value of 1.46 mg/mL. Purification of novel ACE-inhibitory peptides was carried out using ultrafiltration and consecutive chromatographic methods. A novel ACE-inhibitory peptide, with 66-fold increase in ACE-inhibitory activity compared to that of FSE, was isolated from FSE through a five-step purification procedure. The amino acid sequence of the purified ACE-inhibitory peptides was determined to be Leu-Val-Gln-Gly-Ser by Edman degradation method, and its IC₅₀ value was 22 μg/mL (43.7 μM).     

Axenic culture of Gyrodinium impudicum strain KG03, a marine red-tide microalga that produces exopolysaccharide

An exopolysaccharide-producing microalgal dinoflagellate was isolated from a red-tide bloom and designated strain KG03. A bacteria-free culture of strain KG03 was achieved using a modified wash with phototaxis and antibiotic treatment. Combined treatment with neomycin and cephalosporin was the most effective for eliminating the bacteria associated with the microalgae. Strain KG03 was identified as Gyrodinium impudicum by analyzing the ITS regions of the 5.8S rDNA, 18S rDNA, morphological phenotype and fatty acid composition. The exopolysaccharide production and cell growth in a 300-ml photobioreactor were increased 2.7- and 2.4-fold, respectively, compared with that in a flask culture at the first isolation step.     

Growth and tuberization of transgenic potato plants expressing sense and antisense sequences of Cu/Zn superoxide dismutase from lily chloroplasts

Overexpression of a chloroplast-localized Cu/Zn superoxide dismutase (chCu/ZnSOD) obtained from lily significantly affects the growth and shape of potato tubers from anin vitro culture system (Kim et al., 2007). Here, we further characterized the sense and antisense transgenic potatoes grown and pots and the greenhouse to investigate the potential for more practical field applications of such phenotypic manipulations. Underin vitro conditions, antisense transgenic plants showed increased shoot growth, delayed tuberization, and altered tuber shapes. When antisense plants were treated with paclobutrazol, an inhibitor of GA biosynthesis, tuberization efficiency and tuber shape were recovered to a status very similar to that ofin vitro- grown wild-type plants. Our results strongly support the idea that potato tuberization and shape is mediated by SOD-catalyzed reactive oxygen species, possibly via the GA biosynthesis pathway.     

Rapid sexing of preimplantation bovine embryo using consecutive and multiplex polymerase chain reaction (PCR) with biopsied single blastomere.

The objective of this study was to establish a rapid and reliable PCR method for the sexing of 8- to 16-cell stage bovine embryos. The BOV97M and bovine 1.715 satellite DNA sequences were selected for amplification of male- and bovine-specific DNA, respectively. But the unequal number of copies of these two repetitive sequences required some modification of the multiplex PCR method. In consecutive and multiplex PCR, the first 10 PCR cycles were done with male-specific primer followed by an additional 23 cycles with bovine-specific primer. In this PCR method, the appearance of male- and bovine-specific bands was independent of the DNA concentration. This PCR method was applied successfully using groups of 8, 4, 2, and 1 blastomeres dissociated from the embryos, and the sexing efficiency was 100.0, 96.3, 94.3 and 92.1%, respectively. The coincident rate of sex determination between biopsied single blastomere and matched blastocyst was 90.0%. Therefore the developmental potential from 8- to 16-cell stage embryos to the blastocyst stage was not significantly different (P>0.2) for intact embryo (42.3%) than for demi-embryos (53.8%), suggesting that trauma to the demi-embryo caused by single-blastomere aspiration using a bevelled micropipette was very small. In conclusion, we developed a rapid (within 2 hours) and effective PCR method for the sexing of 8- to 16-cell stage bovine embryos using a single blastomere.     

Zinc(II) ion mediates tamoxifen-induced autophagy and cell death in MCF-7 breast cancer cell line

Treatment of MCF-7 cells with tamoxifen induced vacuole formation and cell death. Levels of the autophagy marker, microtubule-associated protein light chain 3 (LC3)-II also increased, and GFP-LC3 accumulated in and around vacuoles in MCF-7 cells exposed to tamoxifen, indicating that autophagy is involved in tamoxifen-induced changes. Live-cell confocal microscopy with FluoZin-3 staining and transmission electron microscopy with autometallographic staining revealed that labile zinc(II) ion (Zn²⁺) accumulated in most acidic LC3(+) autophagic vacuoles (AVs). Chelation of Zn²⁺ with N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) blocked the increase in phospho-Erk and LC3-II levels, and attenuated AV formation and cell death. Conversely, the addition of ZnCl₂ markedly potentiated tamoxifen-induced extracellular signal-regulated kinase (Erk) activation, autophagy and cell death, indicating that Zn²⁺ has an important role in these events. Tamoxifen-induced death was accompanied by increased oxidative stress and lysosomal membrane permeabilization (LMP) represented as release of lysosomal cathepsins into cytosol. Treatment with the antioxidant N-acetyl-l-cysteine (NAC) blunted the increase in Zn²⁺ levels and reduced LC3-II conversion, cathepsin D release and cell death induced by tamoxifen. And cathepsin inhibitors attenuated cell death, indicating that LMP contributes to tamoxifen-induced cell death. Moreover, TPEN blocked tamoxifen-induced cathepsin D release and increase in oxidative stress. The present results indicate that Zn²⁺ contributes to tamoxifen-induced autophagic cell death via increase in oxidative stress and induction of LMP.     

An affinity-based scoring scheme for predicting DNA-binding activities of modularly assembled zinc-finger proteins

Zinc-finger proteins (ZFPs) have long been recognized for their potential to manipulate genetic information because they can be engineered to bind novel DNA targets. Individual zinc-finger domains (ZFDs) bind specific DNA triplet sequences; their apparent modularity has led some groups to propose methods that allow virtually any desired DNA motif to be targeted in vitro. In practice, however, ZFPs engineered using this ‘modular assembly’ approach do not always function well in vivo. Here we report a modular assembly scoring strategy that both identifies combinations of modules least likely to function efficiently in vivo and provides accurate estimates of their relative binding affinities in vitro. Predicted binding affinities for 53 ‘three-finger’ ZFPs, computed based on energy contributions of the constituent modules, were highly correlated (r = 0.80) with activity levels measured in bacterial two-hybrid assays. Moreover, K_d values for seven modularly assembled ZFPs and their intended targets, measured using fluorescence anisotropy, were also highly correlated with predictions (r = 0.91). We propose that success rates for ZFP modular assembly can be significantly improved by exploiting the score-based strategy described here.     

The Roles of Adipokines,Proinflammatory Cytokines,and Adipose Tissue Macrophages in Obesity-Associated Insulin Resistance in Modest Obesity and Early Metabolic Dysfunction

The roles of adipokines, proinflammatory cytokines, and adipose tissue macrophages in obesity-associated insulin resistance have been explored in both animal and human studies. However, our current understanding of obesity-associated insulin resistance relies on studies of artificial metabolic extremes. The purpose of this study was to explore the roles of adipokines, proinflammatory cytokines, and adipose tissue macrophages in human patients with modest obesity and early metabolic dysfunction. We obtained omental adipose tissue and fasting blood samples from 51 females undergoing gynecologic surgery. We investigated serum concentrations of proinflammatory cytokines and adipokines as well as the mRNA expression of proinflammatory and macrophage phenotype markers in visceral adipose tissue using ELISA and quantitative RT-PCR. We measured adipose tissue inflammation and macrophage infiltration using immunohistochemical analysis. Serum levels of adiponectin and leptin were significantly correlated with HOMA-IR and body mass index. The levels of expression of MCP-1 and TNF-α in visceral adipose tissue were also higher in the obese group (body mass index ≥ 25). The expression of mRNA MCP-1 in visceral adipose tissue was positively correlated with body mass index (r = 0.428, p = 0.037) but not with HOMA-IR, whereas TNF-α in visceral adipose tissue was correlated with HOMA-IR (r = 0.462, p = 0.035) but not with body mass index. There was no obvious change in macrophage phenotype or macrophage infiltration in patients with modest obesity or early metabolic dysfunction. Expression of mRNA CD163/CD68 was significantly related to mitochondrial-associated genes and serum inflammatory cytokine levels of resistin and leptin. These results suggest that changes in the production of inflammatory biomolecules precede increased immune cell infiltration and induction of a macrophage phenotype switch in visceral adipose tissue. Furthermore, serum resistin and leptin have specific roles in the regulation of adipose tissue macrophages in patients with modest obesity or early metabolic dysfunction.     

Benzamide derivatives as blockers of Kv1.3 ion channel

The voltage-gated potassium channel, Kv1.3, is present in human T-lymphocytes. Blockade of Kv1.3 results in T-cell depolarization, inhibition of T-cell activation, and attenuation of immune responses in vivo. A class of benzamide Kv1.3 channel inhibitors has been identified. The structure-activity relationship within this class of compounds in two functional assays, Rb_Kv and T-cell proliferation, is presented. In in vitro assays, trans isomers display moderate selectivity for binding to Kv1.3 over other Kv1.x channels present in human brain.