A novel combination of selective agents for isolation of Leptospira species

A novel combination of antimicrobial agents (sulfamethoxazole, 40 μg/mL; trimethoprim, 20 μg/mL; amphotericin B, 5 μg/mL; fosfomycin, 400 μg/mL; and 5-fluorouracil, 100 μg/mL) was developed for selective isolation of leptospires from contaminated samples. The growth of 16 microorganisms considered as possible contaminants during isolation of Leptospira were inhibited by this antimicrobial cocktail. In contrast, the growth of a smaller inoculum (10(1) cells per mL) of 25 Leptospira strains (representing 18 serovars/serogroups of 5 species) was not suppressed by this antimicrobial combination. This cocktail, after being incorporated into Leptospira growth medium (Korthof's), successfully detected leptospires in environmental soil and water. Based on the results, this selective medium has the potential to meet the existing need for an effective selective medium for the isolation of Leptospira.     

Lentiviral vector-mediated gene transfer to endotherial cells compared with adenoviral and retroviral vectors

Human immunodeficiency virus (HIV, lentivirus) vector has attractive features for gene therapy, including the ability to transduce non-dividing cells and long-term transgene expression. We have already reported that lentivirus vector can transduce well-differentiated rat cardiac myocytes. Endothelial cells (EC) are an attractive target for gene therapy, both for the treatment of cardiovascular disease and for the systemic delivery of recombinant gene products directly into the circulation. There are several reports regarding application of adenovirus and retrovirus based vectors to EC. However, there have been few reports which show the effect to lentivirus-mediated gene transfer efficiency, compared with adenovirus and retrovirus. In this study, bovine aortic endothelial cells (BAECs) were infected, in vitro, with these virus vectors. Transduction efficiency (TE) of beta-Gal gene transfer in BAECs by adenovirus, lentivirus, or retrovirus at MOI10 (Multiplicity of infection) (determined on Hela cells) is 69+/-11, 33+/-8, or 22+/-6% respectively. In adenovirus and lentivirus, almost 100% of BAECs were transduced at MOI 50. However, in retrovirus, TE showed only 48+/-6% at MOI 50 and no increase at MOI 100. The percentage of beta-Gal positive cells was decreased rapidly at longer passage of cells after being transduced by adenovirus. However, lentivirus and retrovirus showed sustained higher percentage of positive cells. Furthermore, transduction by lentiviral vectors had no significant effect on viability of BAECs. Our results indicate that lentivirus showed high-level and long term gene expression in BAECs. Lentivirus can be an effective vector for the ex vivo, genetically modified EC implantation and in vivo gene therapy.     

Rice germline‐specific Argonaute MEL1 protein binds to phasiRNAs generated from more than 700 lincRNAs

Small RNAs that interact with Argonaute (AGO) proteins play central roles in RNA‐mediated silencing. MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), a rice AGO, has specific functions in the development of pre‐meiotic germ cells and the progression of meiosis. Here, we show that MEL1, which is located mostly in the cytoplasm of germ cells, associates preferentially with 21‐nucleotide phased small interfering RNAs (phasiRNAs) that bear a 5′‐terminal cytosine. Most phasiRNAs are derived from 1171 intergenic clusters distributed on all rice chromosomes. From these clusters, over 700 large intergenic, non‐coding RNAs (lincRNAs) that contain the consensus sequence complementary to miR2118 are transcribed specifically in inflorescences, and cleaved within the miR2118 site. Cleaved lincRNAs are processed via DICER‐LIKE4 (DCL4) protein, resulting in production of phasiRNAs. This study provides the evidence that the miR2118‐dependent and the DCL4‐dependent pathways are both required for biogenesis of 21‐nt phasiRNAs associated with germline‐specific MEL1 AGO in rice, and over 700 lincRNAs are key factors for induction of this biogenesis during reproductive‐specific stages.     

Distribution of Mammalian-Like Melanopsin in Cyclostome Retinas Exhibiting a Different Extent of Visual Functions

Mammals contain 1 melanopsin (Opn4) gene that is expressed in a subset of retinal ganglion cells to serve as a photopigment involved in non-image-forming vision such as photoentrainment of circadian rhythms. In contrast, most nonmammalian vertebrates possess multiple melanopsins that are distributed in various types of retinal cells; however, their functions remain unclear. We previously found that the lamprey has only 1 type of mammalian-like melanopsin gene, which is similar to that observed in mammals. Here we investigated the molecular properties and localization of melanopsin in the lamprey and other cyclostome hagfish retinas, which contribute to visual functions including image-forming vision and mainly to non-image-forming vision, respectively. We isolated 1 type of mammalian-like melanopsin cDNA from the eyes of each species. We showed that the recombinant lamprey melanopsin was a blue light-sensitive pigment and that both the lamprey and hagfish melanopsins caused light-dependent increases in calcium ion concentration in cultured cells in a manner that was similar to that observed for mammalian melanopsins. We observed that melanopsin was distributed in several types of retinal cells, including horizontal cells and ganglion cells, in the lamprey retina, despite the existence of only 1 melanopsin gene in the lamprey. In contrast, melanopsin was almost specifically distributed to retinal ganglion cells in the hagfish retina. Furthermore, we found that the melanopsin-expressing horizontal cells connected to the rhodopsin-containing short photoreceptor cells in the lamprey. Taken together, our findings suggest that in cyclostomes, the global distribution of melanopsin in retinal cells might not be related to the melanopsin gene number but to the extent of retinal contribution to visual function.     

Aldosterone induces interleukin-18 through endothelin-1, angiotensin II, Rho/Rho-kinase, and PPARs in cardiomyocytes

Aldosterone (Aldo) is recognized as an important risk factor for cardiovascular diseases. IL-18 induces myocardial hypertrophy, loss of contractility of cardiomyocytes, and apoptosis leading myocardial dysfunction. However, so far, there have been few reports concerning the interaction between Aldo and IL-18. The present study examined the effects and mechanisms of Aldo on IL-18 expression and the roles of peroxisome proliferator-activated receptor (PPAR) agonists in rat cardiomyocytes. We used cultured rat neonatal cardiomyocytes stimulated with Aldo to measure IL-18 mRNA and protein expression, Rho-kinase, and NF-kappaB activity. We also investigated the effects of PPAR agonists on these actions. Aldo, endothelin-1 (ET-1), and angiotensin II (ANG II) increased IL-18 mRNA and protein expression. Mineralocorticoid receptor antagonists, endothelin A receptor antagonist, and ANG II receptor antagonist inhibited Aldo-induced IL-18 expression. Aldo induced ET-1 and ANG II production in cultured media. Moreover, Rho/Rho-kinase inhibitor and statin inhibited Aldo-induced IL-18 expression. On the other hand, Aldo upregulated the activities of Rho-kinase and NF-kappaB. PPAR agonists attenuated the Aldo-induced IL-18 expression and NF-kappaB activity but not the Rho-kinase activity. Our findings indicate that Aldo induces IL-18 expression through a mechanism that involves, at a minimum, ET-1 and ANG II acting via the Rho/Rho-kinase and PPAR/NF-kappaB pathway. The induction of IL-18 in cardiomyocytes by Aldo, ET-1, and ANG II might, therefore, cause a deterioration of the cardiac function in an autocrine and paracrine fashion. The inhibition of the IL-18 expression by PPAR agonists might be one of the mechanisms whereby the beneficial cardiovascular effects are exerted.     

Light-induced absorbance changes of carotenoids in brown algae

Light-induced absorbance changes were studied for brown algae with 23 species and a pronounced absorbance change around 563 nm was found in all algae examined. 3-(3,4-dichlorophenyl)-1,1-dimethylurea and gramicidin J suppressed the initial rate and the magnitude of the absorbance change. Carbonylcyanidem-chlorophenylhydrazone did not affect the initial rate but decreased the maximum level of the change. All thalli and the chloroplasts tested had an absorption band at around 540 nm due to fucoxanthin which accounted for about 70–90% of the total carotenoids in brown algae. It is proposed that the 563 nm-change is caused by the red shift of fucoxanthin responding to the light-induced change in the membrane potential of the thylakoid system.