Effects of target sequence and sense versus anti-sense strands on gene correction with single-stranded DNA fragments

The correction of an inactivated hygromycin resistance and enhanced green fluorescent protein (Hyg-EGFP) fusion gene by a several hundred-base single-stranded (ss) DNA fragment has been reported. In this study, the effectiveness of this type of gene correction was examined for various positions in the rpsL gene. Sense and anti-sense ssDNA fragments were prepared, and the gene correction efficiencies were determined by co-introduction of the target plasmid containing the gene with the ssDNA fragments. The gene correction efficiency varied (0.8-9.3%), depending on target positions and sense/anti-sense strands. Sense ssDNA fragments corrected the target gene with equal or higher efficiencies as compared to their anti-sense counterparts. The target positions corrected with high efficiency by the sense fragments also tended to be corrected efficiently by the anti-sense fragments. These results suggest that the sense ssDNA fragments are useful for the correction of mutated genes. The variation in the correction efficiency may depend on the sequence of the target position in double-stranded DNA.     

The second dimer interface of MT1-MMP, the transmembrane domain, is essential for ProMMP-2 activation on the cell surface

Activation of proMMP-2 and cell surface collagenolysis are important activities of membrane-type 1 matrix metalloproteinase (MT1-MMP) to promote cell migration in tissue, and these activities are regulated by homodimerization of MT1-MMP on the cell surface. In this study, we have identified the transmembrane domain as a second dimer interface of MT1-MMP in addition to the previously identified hemopexin domain. Our analyses indicate that these two modes of dimerization have different roles; transmembrane-dependent dimerization is critical for proMMP-2 activation, whereas hemopexin-dependent dimerization is important for degradation of collagen on the cell surface. Our finding provides new insight into the potential molecular arrangement of MT1-MMP contributing to its function on the cell surface.     

Atrial natriuretic peptide and cGMP activate sodium transport through PKA-dependent pathway in the urinary bladder of the Japanese tree frog

The effects of atrial natriuretic peptide (ANP) and cGMP on transepithelial ion transport were examined in the urinary bladder of the Japanese tree frog, Hyla japonica, using Ussing chamber voltage-clamp and whole-cell patch-clamp techniques. When the bladders were exposed to 4.4×10⁻¹¹ to 10⁻⁶ M ANP or 10⁻⁷ to 3×10⁻⁴ M 8-Br-cGMP, both the transepithelial potential difference (PD) and the short-circuit current (Isc) were significantly increased in a concentration-response manner. The cGMP-dependent responses were inhibited in a Na⁺-free bath solution and in the presence of amiloride. The cGMP-dependent increases in Isc were significantly inhibited by specific PKA inhibitors (5×10⁻⁷ M KT-5720 and >10⁻⁵ M H-89), but not by a specific PKG inhibitor (5×10⁻⁷ M KT-5823). ANP-dependent increases in Isc were also significantly inhibited by KT-5720. In the patch-clamp study, ANP and cGMP significantly increased in inward currents involving Na⁺ uptake. These results suggest that a cross-talk mechanism exists between cAMP and cGMP signaling pathways, which leads to Na⁺ transport in the frog urinary bladder. In addition, the cGMP-dependent increases in Isc were partially inhibited by 10⁻⁴ M l-cis-diltiazem, a specific inhibitor of cyclic nucleotide-gated (CNG) channels. These results also suggest a relation between CNG channels and the cGMP-dependent increases in Na⁺ absorption of the frog urinary bladder.     

AKT signaling promotes derivation of embryonic germ cells from primordial germ cells

Primordial germ cells (PGCs) are embryonic germ cell precursors. Although the developmental potency of PGCs is restricted to the germ lineage, PGCs can acquire pluripotency, as verified by the in vitro establishment of embryonic germ (EG) cells and the in vivo production of testicular teratomas. PGC-specific inactivation of PTEN, which is a lipid phosphatase antagonizing phosphoinositide-3 kinase (PI3K), enhances both EG cell production and testicular teratoma formation. Here, we analyzed the effect of the serine/threonine kinase AKT, one of the major downstream effectors of PI3K, on the developmental potency of PGCs. We used transgenic mice that expressed an AKT-MER fusion protein, the kinase activity of which could be regulated by the ligand of modified estrogen receptor (MER), 4-hydroxytamoxifen. We found that hyperactivation of AKT signaling in PGCs at the proliferative phase dramatically augmented the efficiency of EG cell establishment. Furthermore, AKT signaling activation substituted to some extent for the effects of bFGF, an essential growth factor for EG cell establishment. By contrast, AKT activation had no effect on germ cells that were in mitotic arrest or that began meiosis at a later embryonic stage. In the transgenic PGCs, AKT activation induced phosphorylation of GSK3, which inhibits its kinase activity; enhanced the stability and nuclear localization of MDM2; and suppressed p53 phosphorylation, which is required for its activation. The p53 deficiency, but not GSK3 inhibition, recapitulated the effects of AKT hyperactivation on EG cell derivation, suggesting that p53 is one of the crucial downstream targets of the PI3K/AKT signal and that GSK3 is not.     

Ingested beta-carotene enhances glutathione level and up-regulates the activity of cysteine cathepsin in murine splenocytes

To elucidate health benefits of beta-carotene, especially on immunity, we measured redox-related indices in spleen cells from BALB/c mice supplemented with various amounts of beta-carotene. In mice supplemented with beta-carotene in their diet, glutathione, an intracellular anti-oxidation agent, increased in their splenocytes. This change was highly correlated with the accumulation of beta-carotene, but not with that of retinol. The increase in glutathione was accompanied by an increase in mRNA for gamma-glutamylcysteine synthetase, a rate-limiting enzyme for glutathione synthesis. The higher the glutathione content was in the spleen cells, the higher the activity of cysteine cathepsin became in crude antigen-presenting cells contained in the spleen. These data suggest that accumulated beta-carotene in splenocytes, without being metabolized, caused an increase in the intracellular glutathione level, thereby anti-oxidatively supporting the activity of redox-sensitive lysosomal protease, which is involved in antigen-presentation.     

Characterization of plant XRCC1 and its interaction with proliferating cell nuclear antigen

In plants, there are no DNA polymerase β (Pol β) and DNA ligase III (Lig3) genes. Thus, the plant short-patch base excision repair (short-patch BER) pathway must differ considerably from that in mammals. We characterized the rice (Oryza Sativa L. cv. Nipponbare) homologue of the mammalian X-ray repair cross complementing 1 (XRCC1), a well-known BER protein. The plant XRCC1 lacks the N-terminal domain (NTD) which is required for Pol β binding and is essential for mammalian cell survival. The recombinant rice XRCC1 (OsXRCC1) protein binds single-stranded DNA (ssDNA) as well as double-stranded DNA (dsDNA) and also interacts with rice proliferating cell nuclear antigen (OsPCNA) in a pull-down assay. Through immunoprecipitation, we demonstrated that OsXRCC1 forms a complex with PCNA in vivo. OsXRCC1 mRNA was expressed in all rice organs and was induced by application of bleomycin, but not of MMS, H₂O₂ or UV-B. Bleomycin also increased the fraction of OsXRCC1 associated with chromatin. These results suggest that OsXRCC1 contributes to DNA repair pathways that differ from the mammalian BER system.     

Trace and Major Elements Status in Bronchoalveolar Lavage Fluid in Dogs with or without Bronchopneumonia

The aim of this study was to investigate the relationships between the bronchopneumonia and mean concentrations of those trace elements in bronchoalveolar lavage fluid (BALF). Twenty-nine dogs were included this study (17 healthy dogs and 12 dogs with respiratory disease). Each BALF sample had been obtained during bronchoscope examination by use of a standardized method. The concentrations of Al, Br, Ca, Cu, Fe, K, Ni, P, Si, Sr and Zn in BALF were measured by the particle-induced X-ray emission method. We found no relationship between the bronchopneumonia and the levels of elements in the BALF, except Ca, P and Zn. The dogs with respiratory disease were found to have a large amount of Ca and Zn, and a high Ca/P and Zn/Cu ratios in BALF compared to those without respiratory disease.