Liquid-Based Iterative Recombineering Method Tolerant to Counter-Selection Escapes

Selection-based recombineering is a flexible and proven technology to precisely modify bacterial genomes at single base resolution. It consists of two steps of homologous recombination followed by selection/counter-selection. However, the shortage of efficient counter-selectable markers limits the throughput of this method. Additionally, the emergence of ‘selection escapees’ can affect recombinant pools generated through this method, and they must be manually removed at each step of selection-based recombineering. Here, we report a series of efforts to improve the throughput and robustness of selection-based recombineering and to achieve seamless and automatable genome engineering. Using the nucleoside kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the non-natural nucleoside dP, a highly efficient, rapid, and liquid-based counter-selection system was established. By duplicating hsvtk gene, combined with careful control of the population size for the subsequent round, we effectively eliminated selection escapes, enabling seamless and multiple insertions/replacement of gene-size fragments in the chromosome. Four rounds of recombineering could thus be completed in 10 days, requiring only liquid handling and without any need for colony isolation or genotype confirmation. The simplicity and robustness of our method make it broadly accessible for multi-locus chromosomal modifications.     

Roles of adjoining Asp and Cys residues of first matrix-facing loop in transport activity of yeast and bovine mitochondrial ADP/ATP carriers

The mitochondrial ADP/ATP carrier (AAC) transports substrate by interconversion of its conformation between m- and c-states. The 1st loop facing the matrix (LM1) is extruded into the matrix in the m-state and is suggested to intrude into the mitochondrial membrane on conversion to the c-state conformation [Hashimoto, M., Majima, E., Goto, S., Shinohara, Y., and Terada, H. (1999) Biochemistry 38, 1050-1056]. To elucidate the mechanism of the translocation of LM1, we examined the effects of site-directed mutagenesis of two adjoining residues, Cys56 and Asp55 in the bovine type 1 AAC and Cys73 and Asp72 in the yeast type 2 AAC, on the substrate transport activity. We found that (i) replacement of the Cys by bulky and hydrophilic residues was unfavorable for efficient transport activity, (ii) the carboxyl groups of the Asp residues of the bovine and yeast AACs were essential and strictly position-specific, and (iii) hence, the mutation to Glu showed transport activity comparable to that of the native AACs. Based on these results, we discussed the functional role of LM1 in the transport activity of AAC.     

Further characterization of loss of heterozygosity enhanced by p53 abrogation in human lymphoblastoid TK6 cells: disappearance of endpoint hotspots

Loss of heterozygosity (LOH) is the predominant mechanism of spontaneous mutagenesis at the heterozygous thymindine kinase locus (tk) in TK6 cells. LOH events detected in spontaneous TK⁻ mutants (110 clones from p53 wild-type cells TK6-20C and 117 clones from p53-abrogated cells TK6-E6) were analyzed using 13 microsatellite markers spanning the whole of chromosome 17. Our analysis indicated an approximately 60-fold higher frequency of terminal deletions in p53-abrogated cells TK6-E6 compared to p53 wild-type cells TK6-20C whereas frequencies of point mutations (non-LOH events), interstitial deletions, and crossing over events were found to increase only less than twofold by such p53 abrogation. We then made use of an additional 17 microsatellite markers which provided an average map-interval of 1.6 Mb to map various LOH endpoints on the 45 Mb portion of chromosome 17q corresponding to the maximum length of LOH tracts (i.e. from the distal marker D17S932 to the terminal end). There appeared to be four prominent peaks (I–IV) in the distribution of LOH endpoints/Mb of Tk6-20C cells that were not evident in p53-abrogated cells TK6-E6, where they appeared to be rather broadly distributed along the 15–20 Mb length (D17S1807 to D17S1607) surrounding two of the peaks that we detected in TK6-20C cells (peaks II and III). We suggest that the chromosomal instability that is so evident in TK6-E6 cells may be due to DNA double-strand break repair occurring through non homologous end-joining rather than allelic recombination.     

Possible association of Neu2 with plasma membrane fraction from mouse thymus exhibited sialidase activity with fetuin at pH 7.0 but not at pH 4.5

Compared to other organs, the mouse thymus exhibits a high level of sialidase activity in both the soluble and crude membrane fractions, as measured at neutral pH using 4MU‐Neu5Ac as a substrate. The main purpose of the present study was to identify the sialidase with a high level of the activity at neutral pH in the crude membrane. Several parameters were analyzed using the soluble (S) fraction, N and D fractions that were obtained by NP‐40 or DOC/NP‐40 solubilization from the thymus crude membrane. The main sialidase activity in the N fraction exhibited almost the same pI as that of soluble Neu2 and 60% of the activity was removed from the membrane by three washes with 10 mM Tris‐buffer, at pH 7.0. The N fraction preferentially hydrolyzed the sialic acid bond of glycoprotein and exhibited sialidase activity with fetuin at pH 7.0 but not at pH 4.5. The same activity was observed in a plasma membrane‐rich fraction. To date, the removal of sialic acid from fetuin at pH 7.0 was reported only with soluble Neu2 and the membrane fraction from Neu2‐transfected COS cells. We analyzed the gene that controls the sialidase activity in the crude membrane fraction at pH 7.0 using SMXA recombinant mice and found that compared with other three genes, Neu2 presented the best correlation with the activity level. We suggest that Neu2 is most likely responsible for the main activity in the N fraction, due to its association with the membrane by an unknown mechanism.     

Deletion of Hypoxia-Inducible Factor-1α in Adipocytes Enhances Glucagon-Like Peptide-1 Secretion and Reduces Adipose Tissue Inflammation

It is known that obese adipose tissues are hypoxic and express hypoxia-inducible factor (HIF)-1α. Although some studies have shown that the expression of HIF-1α in adipocytes induces glucose intolerance, the mechanisms are still not clear. In this study, we examined its effects on the development of type 2 diabetes by using adipocyte-specific HIF-1α knockout (ahKO) mice. ahKO mice showed improved glucose tolerance compared with wild type (WT) mice. Macrophage infiltration and mRNA levels of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor α (TNFα) were decreased in the epididymal adipose tissues of high fat diet induced obese ahKO mice. The results indicated that the obesity-induced adipose tissue inflammation was suppressed in ahKO mice. In addition, in the ahKO mice, serum insulin levels were increased under the free-feeding but not the fasting condition, indicating that postprandial insulin secretion was enhanced. Serum glucagon-like peptide-1 (GLP-1) levels were also increased in the ahKO mice. Interestingly, adiponectin, whose serum levels were increased in the obese ahKO mice compared with the obese WT mice, stimulated GLP-1 secretion from cultured intestinal L cells. Therefore, insulin secretion may have been enhanced through the adiponectin-GLP-1 pathway in the ahKO mice. Our results suggest that the deletion of HIF-1α in adipocytes improves glucose tolerance by enhancing insulin secretion through the GLP-1 pathway and by reducing macrophage infiltration and inflammation in adipose tissue.     

Acute and Temporal Expression of Tumor Necrosis Factor (TNF)-α-stimulated Gene 6 Product,TSG6, in Mesenchymal Stem Cells Creates Microenvironments Required for Their Successful Transplantation into Muscle Tissue

Previously, we demonstrated that when mesenchymal stem cells (MSCs) from mouse ES cells were transplanted into skeletal muscle, more than 60% of them differentiated into muscles in the crush-injured tibialis anterior muscle in vivo, although MSCs neither differentiated nor settled in the intact muscle. Microenvironments, including the extracellular matrix between the injured and intact muscle, were quite different. In the injured muscle, hyaluronan (HA), heavy chains of inter-α-inhibitor (IαI), CD44, and TNF-α-stimulated gene 6 product (TSG-6) increased 24–48 h after injury, although basement membrane components of differentiated muscle such as perlecan, laminin, and type IV collagen increased gradually 4 days after the crush. We then investigated the microenvironments crucial for cell transplantation, using the lysate of C2C12 myotubules for mimicking injured circumstances in vivo. MSCs settled in the intact muscle when they were transplanted together with the C2C12 lysate or TSG6. MSCs produced and released TSG6 when they were cultured with C2C12 lysates in vitro. MSCs pretreated with the lysate also settled in the intact muscle. Furthermore, MSCs whose TSG6 was knocked down by shRNA, even if transplanted or pretreated with the lysate, could not settle in the muscle. Immunofluorescent staining showed that HA and IαI always co-localized or were distributed closely, suggesting formation of covalent complexes, i.e. the SHAP-HA complex in the presence of TSG6. Thus, TSG6, HA, and IαI were crucial factors for the settlement and probably the subsequent differentiation of MSCs.     

Mesenchymal stem cells originating from ES cells show high telomerase activity and therapeutic benefits

We establish a novel method for the induction and collection of mesenchymal stem cells using a typical cell surface marker, CD105, through adipogenesis from mouse ES cells. ES cells were cultured in a medium for adipogenesis. Mesenchymal stem cells from mouse ES cells were easily identified by the expression of CD105, and were isolated and differentiated into multiple mesenchymal cell types. Mesenchymal stem cells showed remarkable telomerase activity and sustained their growth for a long time with a high potential for differentiation involving skeletal myogenesis in vitro. When mesenchymal stem cells were transplanted into the injured tibialis anterior muscles, they differentiated into skeletal muscle cells in vivo. In addition, they improved the vascular formation, but never formed teratoma for longer than 6 months. Gene expression profiles revealed that mesenchymal stem cells lost pluripotency, while they acquired high potential to differentiate into mesenchymal cell lines. They thus indicate a promising new source of cell-based therapy without teratoma formation.     

Alternative Fas-mediated cell death pathway is dependent on the different cleavage patterns of procaspase-8

Molecular and Cellular Biochemistry - It has been reported that mitochondria-independent or mitochondria-dependent (type I/II) Fas signaling pathways in leukemia cells depend on the amount of...     

Heparin Cofactor II,a Serine Protease Inhibitor,Promotes Angiogenesis via Activation of the AMP-activated Protein Kinase-Endothelial Nitric-oxide Synthase Signaling Pathway

We previously clarified that heparin cofactor II (HCII), a serine proteinase inhibitor, exerts various protective actions on cardiovascular diseases in both experimental and clinical studies. In the present study, we aimed to clarify whether HCII participates in the regulation of angiogenesis. Male heterozygous HCII-deficient (HCII^+/−) mice and male littermate wild-type (HCII^+/+) mice at the age of 12–16 weeks were subjected to unilateral hindlimb ligation surgery. Laser speckle blood flow analysis showed that blood flow recovery in response to hindlimb ischemia was delayed in HCII^+/− mice compared with that in HCII^+/+ mice. Capillary number, arteriole number, and endothelial nitric-oxide synthase (eNOS), AMP-activated protein kinase (AMPK), and liver kinase B1 (LKB1) phosphorylation in ischemic muscles were decreased in HCII^+/− mice. Human purified HCII (h-HCII) administration almost restored blood flow recovery, capillary density, and arteriole number as well as phosphorylation levels of eNOS, AMPK, and LKB1 in ischemic muscles of HCII^+/− mice. Although treatment with h-HCII increased phosphorylation levels of eNOS, AMPK, and LKB1 in human aortic endothelial cells (HAECs), the h-HCII-induced eNOS phosphorylation was abolished by compound C, an AMPK inhibitor, and by AMPK siRNA. In a similar fashion, tube formation, proliferation, and migration of HAECs were also promoted by h-HCII treatment and were abrogated by pretreatment with compound C. HCII potentiates the activation of vascular endothelial cells and the promotion of angiogenesis in response to hindlimb ischemia via an AMPK-eNOS signaling pathway. These findings suggest that HCII is a novel therapeutic target for treatment of patients with peripheral circulation insufficiency.