Inhibition of alpha-globin gene expression by RNAi

RNA interference (RNAi), a process by which target messenger RNA (mRNA) is cleaved by small interfering complementary RNA (siRNA), is widely used for investigations of regulation of gene expression in various cells. In this study, siRNA complementary to 5′ region of exon II of α-globin mRNA was examined for its role in erythroid colony forming cells (ECFCs) isolated from normal peripheral blood donor. On day 6 of cell culture, 1 × 10⁶ ECFCs were transfected with lipofectamine-containing α-globin specific siRNA. After 48 h of transfection, α-globin specific siRNA produced significantly reduction of α-globin mRNA level in a dose-dependent manner, but it did not affect the level of β-globin mRNA. Significantly, decreased numbers of hemoglobinized erythroid cells relative to the control were observed supporting the inhibitory effect of this α-globin mRNA specific siRNA.     

Expression of small RNAs of Bordetella pertussis colonizing murine tracheas

We performed RNA sequencing on Bordetella pertussis, the causative agent of whooping cough, and identified nine novel small RNAs (sRNAs) that were transcribed during the bacterial colonization of murine tracheas. Among them, four sRNAs were more strongly expressed in vivo than in vitro. Moreover, the expression of eight sRNAs was not regulated by the BvgAS two-component system, which is the master regulator for the expression of genes contributing to the bacterial infection. The present results suggest a BvgAS-independent gene regulatory system involving the sRNAs that is active during B. pertussis infection.     

Tissue culture inHaworthia

Effects of three auxins and kinetin on growth of the calluses of two species ofHaworthia, H. aristala andH. setata, were investigated. Stock calluses derived from the flower buds of these species were maintained for two years on RM-1964 agar medium containing 5 mg/l NAA. Small pieces of the stock calluses were transferred to the basal medium containing either auxin, IAA, NAA or 2,4-D in six different concentrations (0.1–50 mg/l) combined with three concentrations (0–2 mg/l) of kinetin; in total, 54 kinds of media were used. Fresh weight of the calluses was measured 0 to 30 days from transfer and transformed to the natural logarithm. The linearity of their growth curves against the culture period was tested. The growth curves of theH. aristata calluses grown in dark and under continuous light and that of theH. setata callus grown in dark gave similar regression coefficients of 0.07 to 0.11, indicating that the doubling time of the callus mass was about 6.3 to 10.1 days. After 42 to 50 days from inoculation, the fresh weight of each individual callus was recorded, and the data were statistically analyzed. All auxins at the concentration of 50 mg/l significantly inhibited callus growth. Kinetin did not affect growth of theH. aristata callus in dark, while its effect on theH. setata callus was detected under light. Interaction of kinetin was found with IAA and 2,4-D inH. aristata and with IAA and NAA inH. setata. REsponses of theH. aristata callus to auxins and kinetin, when grown in dark, were different in several points from those of theH. setata callus grown under light. The best callus growth was observed in the following media; 0.2 mg/l kinetin supplemented with 1 mg/l IAA, or 0.5 mg/l 2,4-D, and 2 mg/l kinetin with 0.5 mg/l NAA inH. aristata, and 0.2 mg/l kinetin supplemented with 1 mg/l IAA, 5 mg/l NAA or 0.1 mg/l 2,4-D inH. setata. Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 413.     

Status of Bone Allografting in Japan – Nation-Wide Survey of Bone Grafting Performed from 1995 through 1999

We report the status of bone allografting in Japan on the basis of the information obtained through questionnaires performed by the Japanese Orthopaedic Association (JOA). JOA performed a nation-wide survey in 2000, in order to clarify the current status of musculoskeletal tissue grafting in the orthopaedic practices in Japan. Conducted period was for 5 years from 1995 to 1999. As the results of this survey, it had been clarified that 92,984 bone graftings, which included autografts, allografts and synthetic bone substitutes, were performed during conducted 5 years. While the allografts were used only in 3,212 cases (3%), autograftings were performed in 64,193 cases (69%), synthetic bone substitutes were used in 25,576 cases (28%) in this series. The proportion of the number of operations for use bone substitutes increased every year, whereas that autografting decreased. The proportion of the number of allografting remained almost unaltered. Of the 706 institutions which answered to have experiences of tissue grafting, only 193 (27%) performed allograft.Since Kitasato University Hospital Bone Bank was developed in 1971, we have applied to clinical while doing basic research for preserved bone allograft. When extensive bone graft is required, allograft is very useful. In Japan, however, allograft is not performed widely. The foundation of regional bone banks is expected to resolve this problem. Since excision of bone preparations from cadaver donors is not common, bone allografts are not supplied sufficiently at present. It is needed to develop a network connecting bone banks in Japan. The enlightenment activities to the ordinary people and medical institutions will also be required.     

M-LP,Mpv17-like protein,has a peroxisomal membrane targeting signal comprising a transmembrane domain and a positively charged loop and up-regulates expression of the manganese superoxide dismutase gene

M-LP (Mpv17-like protein) has been identified as a new protein that has high sequence homology with Mpv17 protein, a peroxisomal membrane protein involved in the development of early onset glomerulosclerosis. In this study, we verified the peroxisomal localization of M-LP by performing dual-color confocal analysis of COS-7 cells cotransfected with green fluorescent protein-tagged M-LP and DsRED2-PTS1, a red fluorescent peroxisomal marker. To characterize the peroxisomal membrane targeting signal, we examined the intracellular localizations of several green fluorescent protein-tagged deletion mutants and demonstrated that, of the three transmembrane segments predicted, the first near the NH(2) terminus and NH(2)-terminal half of the following loop region, which is abundant in positively charged amino acids, were necessary and sufficient for peroxisomal targeting. To elucidate the function of M-LP, we examined the activities of several enzymes involved in reactive oxygen species metabolism in COS-7 cells and found that transfection with M-LP increased the superoxide dismutase activity significantly. Quantitative real-time PCR analysis revealed that the manganese SOD (SOD2) mRNA level of COS-7 cells transfected with M-LP was elevated. These results indicate that M-LP participates in reactive oxygen species metabolism.     

Alternative splicing variants of the human DBL (MCF-2) proto-oncogene

The DBL (MCF-2) proto-oncogene is a prototype guanine nucleotide exchange factor (GEF) that modulates the Rho family of GTPases. In this communication we describe the isolation of three novel splicing variants of Dbl. The prototype Dbl gene (designated var.1 here) contains 25 exons, while splicing variant 2 (var.2) lacks exons 23 and 24. Var.3 contains additional 3 exons from 5(')-UTR in place of exon 1, while var.4, var.2, and var.3 contain a 48bp insertion between exons 10 and 11, resulting in the insertion of 16 amino acids. We found that var.1 was expressed only in brain, whereas var.3 was expressed in heart, kidney, spleen, liver, and testis, and var.4 in brain, heart, kidney, testis, placenta, stomach, and peripheral blood. The Dbl protein was detectable in brain, heart, kidney, intestine, muscle, lung, and testis. An assay for GEF activity revealed that the var.2 exhibits decreased GEF activity towards Cdc42, var.3 exhibits a weak but significant activity toward Rac1 and Cdc42, var.4 exhibits significant activity toward RhoA and Cdc42, while var.1 exhibits no activity toward RhoA, Rac1, or Cdc42. In summary, we describe 4 splicing variants of the human DBL proto-oncogene that show different tissue distributions and GEF specificities.     

Biosynthesis of steroids in ovarian follicles of red seabream,Pagrus major (Sparidae,Teleostei) during final oocyte maturation and the relative effectiveness of steroid metabolites for germinal vesicle breakdown in vitro

The steroid synthesis pathway in the ovarian follicles of the red seabream during final oocyte maturation (FOM) was investigated by incubating intact follicles with different radioactively labeled steroid precursors. During FOM, the steroidogenic shift from estradiol-17beta to 20 beta-hydroxylated progestin production occurred mainly due to a combination of inactivation of C 1720-lyase and activation of 20 beta-hydroxysteroid dehydrogenase. Of the steroids produced, 1720 beta-dihydroxy-4-pregnen-3-one (1720 beta-P) and 1720 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) exhibited the greatest effect on germinal vesicle breakdown (GVBD) in vitro. 1720 beta-P was further converted to its 5 beta-reduced form, 1720 beta-dihydroxy-5 beta-pregnan-3-one (1720 beta-P-5 beta), which had lower GVBD activity, suggesting that 5 beta-reduction plays a role in the inactivation of the maturation-inducing ability of 1720 beta-P. In contrast, no 5 beta-reduced metabolite of 20 beta-S was found. Serum levels of 1720 beta-P and 20 beta-S, measured by ELISA, showed that circulating levels of both progestins increased during FOM, and 20 beta-S levels were approximately twice as high as 1720 beta-P levels. This study clarified the complete steroidogenesis pathway during FOM in red seabream ovarian follicles and showed that two 20 beta-hydroxylated progestins, 1720 beta-P and 20 beta-S, act as maturation-inducing hormones in this species. The catabolites of these two progestins and their physiological roles in reproduction are also discussed.     

Selenomethionine incorporation into a protein by cell-free synthesis

Multi-wavelength anomalous diffraction phasing is especially useful for high-throughput structure determinations. Selenomethionine substituted proteins are commonly used for this purpose. However, the cytotoxicity of selenomethionine drastically reduces the efficiency of its incorporation in in vivo expression systems. In the present study, an improved E. coli cell-free protein synthesis system was used to incorporate selenomethionine into a protein, so that highly efficient incorporation could be achieved. A milligram quantity of selenomethionine-containing Ras was obtained using the cell-free system with dialysis. The mass spectrometry analysis showed that more than 95% of the methionine residues were substituted with selenomethionine. The crystal of this protein grew under the same conditions and had the same unit cell constants as those of the native Ras protein. The three-dimensional structure of this protein, determined by multi-wavelength anomalous diffraction phasing, was almost the same as that of the Ras protein prepared by in vivo expression. Therefore, the cell-free synthesis system could become a powerful protein expression method for high-throughput structure determinations by X-ray crystallography.     

Ability of ferric nitrilotriacetate complex with three pH-dependent conformations to induce lipid peroxidation

This study examined the generation of reactive oxygen species (ROS) and the induction of lipid peroxidation by carcinogenic iron(III)-NTA complex (1:1), which has three conformations with two pKa values (pKa1 approximately 4, pKa2 approximately 8). These conformations are type (a) in acidic conditions of pH 1-6, type (n) in neutral conditions of pH 3-9, and type (b) in basic conditions of pH 7-10. The iron(III)-NTA complex was reduced to iron(II) complex under cool-white fluorescent light without the presence of any reducer. The reduction rates of three species of iron(III)-NTA were in the order type (a) > type (n) > type (b). Iron(III)-NTA-dependent lipid peroxidation was induced in the presence and absence of preformed lipid peroxides (L-OOH) through processes associated with and without photoreduction of iron(III). The order of the abilities of the three species of iron(III)-NTA to initiate the three mechanisms of lipid peroxidation was: (1) type (a) > type (n) > type (b) in lipid peroxidation that is induced L-OOH- and H2O2-dependently and mediated by the photoreduction of iron(III); (2) type (b) > type (n) > type (a) in lipid peroxidation that is induced L-OOH- and H2O2-dependently but not mediated by the photoreduction of iron(III); (3) type (n) > type (b) > type (a) in lipid peroxidation that is induced peroxide-independently and mediated by the photoactivation but not by the photoreduction of iron(III). The rate of lipid peroxidation induced L-OOH-dependently is faster than that induced H2O2-dependently in the mechanism (1), but the rate of lipid peroxidation induced H2O2-dependently is faster than that induced L-OOH-dependently in the mechanism (2). In the lag process of mechanism (3), L-OOH and/or some free radical species, not 1O2, were generated by photoactivation of iron(III)-NTA. These multiple pro-oxidant properties that depend on the species of iron(III)-NTA were postulated to be a principal cause of its carcinogenicity.