Analysis of gene-trap Ds rice populations in Korea

Insertional mutagen-mediated gene tagging populations have been essential resources for analyzing the function of plant genes. In rice, maize transposable elements have been successfully utilized to produce transposant populations. However, many generations and substantial field space are required to obtain a sufficiently sized transposant population. In rice, the japonica and indica subspecies are phenotypically and genetically divergent. Here, callus cultures with seeds carrying Ac and Ds were used to produce 89,700 lines of Dongjin, a japonica cultivar, and 6,200 lines of MGRI079, whose genome is composed of a mixture of the genetic backgrounds of japonica and indica. Of the more than 3,000 lines examined, 67% had Ds elements. Among the Ds-carrying lines, 81% of Dongjin and 63% of MGRI079 contained transposed Ds, with an average of around 2.0 copies. By examining more than 15,000 lines, it was found that 12% expressed the reporter gene GUS during the early-seedling stage. GUS was expressed in root hairs and crown root initials at estimated frequencies of 0.78% and 0.34%, respectively. The 5,271 analyzed Ds loci were found to be randomly distributed over all of the rice chromosomes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sung Han Park, Nam Soo Jun, Chul Min Kim are contributed equally to this paper     

A Ds-insertion mutant of OSH6 (Oryza sativa Homeobox 6) exhibits outgrowth of vestigial leaf-like structures, bracts, in rice

OSH6 (Oryza sativa Homeobox6) is an ortholog of lg3 (Liguleless3) in maize. We generated a novel allele, termed OSH6-Ds, by inserting a defective Ds element into the third exon of OSH6, which resulted in a truncated OSH6 mRNA. The truncated mRNA was expressed ectopically in leaf tissues and encoded the N-terminal region of OSH6, which includes the KNOX1 and partial KNOX2 subdomains. This recessive mutant showed outgrowth of bracts or produced leaves at the basal node of the panicle. These phenotypes distinguished it from the OSH6 transgene whose ectopic expression led to a “blade to sheath transformation” phenotype at the midrib region of leaves, similar to that seen in dominant Lg3 mutants. Expression of a similar truncated OSH6 cDNA from the 35S promoter (35S::ΔOSH6) confirmed that the ectopic expression of this product was responsible for the aberrant bract development. These data suggest that OSH6-Ds interferes with a developmental mechanism involved in bract differentiation, especially at the basal nodes of panicles. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Murine norovirus increases atherosclerotic lesion size and macrophages in Ldlr(-/-) mice

Murine norovirus (MNV) is prevalent in rodent facilities in the United States. Because MNV has a tropism for macrophages and dendritic cells, we hypothesized that it may alter phenotypes of murine models of inflammatory diseases, such as obesity and atherosclerosis. We examined whether MNV infection influences phenotypes associated with diet-induced obesity and atherosclerosis by using Ldlr(-/-) mice. Male Ldlr(-/-) mice were maintained on either a diabetogenic or high-fat diet for 16 wk, inoculated with either MNV or vehicle, and monitored for changes in body weight, blood glucose, glucose tolerance, and insulin sensitivity. Influence of MNV on atherosclerosis was analyzed by determining aortic sinus lesion area. Under both dietary regimens, MNV-infected and control mice gained similar amounts of weight and developed similar degrees of insulin resistance. However, MNV infection was associated with significant increases in aortic sinus lesion area and macrophage content in Ldlr(-/-) mice fed a high-fat diet but not those fed a diabetogenic diet. In conclusion, MNV infection exacerbates atherosclerosis in Ldlr(-/-) mice fed a high-fat diet but does not influence obesity- and diabetes-related phenotypes. Increased lesion size was associated with increased macrophages, suggesting that MNV may influence macrophage activation or accumulation in the lesion area.     

Local kallikrein�Ckinin system is involved in podocyte apoptosis under diabetic conditions

The kallikrein-kinin system (KKS) serves as the physiologic counterbalance to the renin-angiotensin system. This study was conducted to examine the changes in the expression of KKS components in podocytes under diabetic conditions and to elucidate the functional role of bradykinin (BK) in diabetes-associated podocyte apoptosis. Thirty-two rats were injected with either diluent (n = 16, C) or with streptozotocin intraperitoneally (n = 16, DM), and 8 rats from each group were treated with BK infusion for 6 weeks. Immortalized mouse podocytes were cultured in media containing 5.6 mmol/l glucose (NG), NG + 10(-7) mol/l AII (AII), or 30 mmol/l glucose (HG) with or without 10(-8) mol/l BK. Urinary albumin excretion was significantly higher in DM rats, and this increase was ameliorated by BK. Not only kininogen, kallikrein, and BK B1- and B2-receptor expression but also BK levels were significantly decreased in DM glomeruli and in cultured podocytes exposed to HG. The changes in the expressions of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli as well as in HG- and AII-stimulated podocytes were significantly abrogated by BK. The suppressed KSS within podocytes under diabetic condition was associated with podocyte apoptosis, suggesting that BK may be beneficial in preventing podocyte loss in diabetic nephropathy.     

Discrimination of Kalopanax pictus from its varieties and other ‘Kalopanacis Cortex’ plants by multiplex polymerase chain reaction (PCR)

‘Kalopanacis Cortex’ (KC) is an important medicinal material prescribed in Korea for the treatment of various ailments such as paralysis, arthritis, and rheumatism. In Korea, KC is defined as the dried stem bark of Kalopanax pictus. However, the stem barks of Zanthoxylum ailanthoides and Erythrina plants such as E. variegata have also been described as KC and prescribed for the same diseases. Since the pharmacological activities and contents of compounds isolated from the stem barks of these KC plants are obviously different, KC from K. pictus should be clearly discriminated from other stem bark in the best interest of public health. This study sequenced the internal transcribed spacers (ITS) of 52 samples of the KC or KC plants collected from Korea, China, and Vietnam. On the basis of different nucleotide sequences of specific ITS regions among the KC plants, the primer set KP F2/KC R1 was designed to amplify a 398-bp DNA marker for discriminating K. pictus from its varieties and from other KC plants. This primer set, along with the primer sets ZR F1/ZR R4, KP F1/EV R2, and KPF1/KC R1, was successfully amplified with the DNA markers of the Z. ailanthoides, E. variegata, and K. pictus taxa, respectively. The multiplex polymerase chain reaction method developed here not only discriminates the stem bark of K. pictus from stem barks of other KC plants but also identifies KC plants that supply KC in a single process.     

Interrelation between long-chain fatty acid oxidation rate and carnitine palmitoyltransferase 1 activity with different isoforms in rat tissues

This study examined the interrelation between the long-chain fatty acid (LCFA) oxidation rate and the carnitine palmitoyltransferase (CPT) 1 activity in various tissues containing L-CPT1 or M-CPT1. The Liver, kidney, heart, white and red gastrocnemius muscles, and white and brown adipose tissues obtained from Sprague-Dawley rats were examined. In the tissues containing L-CPT1 the liver showed a significantly higher (P<0.01) palmitate oxidation rate and CPT1 activity than the kidney. Among the tissues containing M-CPT1, the brown adipose tissue showed the highest palmitate oxidation rate and CPT1 activity. The tissues containing M-CPT1 (r2=0.907, p<0.001) showed a strong positive correlation between the palmitate oxidation rate and the CPT1 activity. The ratios of the palmitate oxidation rate to the CPT1 activity were calculated. The ratio in the liver was highest and the ratio in the kidney was lowest among the tissues. The ratios of the tissues containing M-CPT1 were similar. These results showed that the LCFA oxidation rates in the tissues containing M-CPT1 were directly proportional to the CPT1 activity, but not similarly proportional to the CPT1 activity in the tissues containing L-CPT1. In conclusion, CPT1 activity seems very important factor for LCFA oxidation, but it might be not the only rate-limiting step in LCFA oxidation.     

Synthesis and evaluation of benzoxazole derivatives as 5-lipoxygenase inhibitors

5-Lipoxygenase (5-LOX) is important enzyme in the biosynthesis of leukotrienes, and is a potential target in the treatment of asthma and allergy. We designed and synthesized a series of benzoxazoles and benzothiazoles as 5-LOX inhibitors. Fourteen compounds prepared showed the inhibition of LTC4 formation with IC₅₀ value of 0.12–23.88 μM. Also two compounds 2d and 2g showed improved airway hypersensitiveness.     

Thermogelling aqueous solutions of alternating multiblock copolymers of poly(L-lactic acid) and poly(ethylene glycol)

We are reporting alternating multiblock copolymers of poly(L-lactic acid)/poly(ethylene glycol) aqueous solution (> 15 wt %) undergoing sol-gel-sol transition as the temperature increases from 20 to 60 degrees C. Micelles of the multiblock copolymers (in water) are about 20 nm in radius at low temperature. They are aggregated to a larger size as the temperature increases, which should play a critical role in the sol-to-gel transition. The transition temperature and gel window were affected by the molecular weight and composition of the multiblock copolymer. In particular, the aqueous solution of an alternating multiblock copolymer (Mn approximately 6700 daltons) prepared from poly(ethylene glycol) (Mn approximately 600 daltons) and poly(L-lactic acid) (Mn approximately 1300 daltons) showed a maximum modulus at body temperature (37 degrees C). The in situ gel forming ability of the polymer aqueous solution in vivo as well as in vitro indicates that it can be a promising injectable biomaterial.     

Doxorubicin induces the persistent activation of intracellular transglutaminase 2 that protects from cell death

The activation of transglutaminase 2 (TG2), an enzyme that catalyzes post-translational modifications of proteins, has been implicated in apoptosis, cell adhesion and inflammatory responses. We previously reported that intracellular TG2 is activated under oxidative stress conditions, such as ultraviolet irradiation, ischemia-reperfusion, and hypoxia. In this study, we examined the effect of genotoxic stress on the intracellular activity of TG2 using doxorubicin which generates reactive oxygen species that lead to double-strand breakage of DNA. We demonstrated that doxorubicin elicits the persistent activation of TG2. Doxorubicin-induced TG2 activity was suppressed by treatment with caffeine at the early phase, N-acetylcysteine at the mid-phase, and EGTA at the late phase. However, treatment with a blocking antibody against TGFβ or toll-like receptor 2 showed no effect on TG2 activity, indicating that at least three different signaling pathways may be involved in the process of TG2 activation. In addition, using MEF cells defective for TG2 and cells overexpressing an activesite mutant of TG2, we revealed that doxorubicin-induced cell death is inversely correlated with TG2 activity. Our findings indicate that the persistent activation of TG2 by doxorubicin contributes to cell survival, suggesting that the mechanism-based inhibition of TG2 may be a novel strategy to prevent drug-resistance in doxorubicin treatment.