Effects of phytosulfokine-α on growth and tropane alkaloid production in transformed roots of Atropa belladonna

Phytosulfokine (PSK)- is a sulphated pentapeptide, isolated fromthe medium of cultured Asparagus officinalis mesophyllcells, that promotes cell proliferation. It is a putative key factor inconditioned medium required for the growth of low-density plant cell cultures.The present study investigates the effect of PSK- on growth and tropanealkaloid production in Atropa belladonna hairy rootstransformed with Agrobacterium rhizogenes (MAFF 03-01724). Although the growth rates ofhairy roots cultured in medium with orwithout PSK- for 4 weeks did not show any differences, the productivityof tropane alkaloids, especially of hyoscyamine, was enhanced by10^–7 or 10^–8 M PSK-. Inaddition, the content of tropane alkaloids in transformed roots treated withPSK- was 1.4 times higher than that of untreated roots after 4 weeks ofculture. The time course of growth and tropane alkaloid production inAtropa belladonna transformed roots suggested thatPSK- influenced the growth of transformed roots during the activegrowingphase, but not tropane alkaloid production.     

Characterization and imprinting status of OBPH1/Obph1 gene: implications for an extended imprinting domain in human and mouse

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.     

Possible relationship of PFGE patterns of Moraxella catarrhalis between hospital- and community-acquired respiratory infections in a community hospital

We describe a prospective study of molecular analysis of Moraxella catarrhalis isolated from a community hospital. Our study was designed to investigate the possible relationship of pulsed-field gel electrophoresis (PFGE) patterns of M. catarrhalis between hospital- and community-acquired respiratory infections. A nosocomial outbreak of M. catarrhalis was observed between September 2000 and September 2001. During the study period, 40 strains of M. catarrhalis were isolated from a total of 32 patients with respiratory infections (26 strains from 18 inpatients, and 14 strains from 14 outpatients). We compared the PFGE patterns in 40 strains of M. catarrhalis isolated from the respiratory tract of the study patients. The genomic types of M. catarrhalis were classified into three PFGE patterns (A, B, and C). Interestingly, the nosocomial outbreak of M. catarrhalis included two patterns (A and B). Of the three patterns, two patterns (A and B) were found in both inpatients and outpatients. More interestingly, two subtypes of pattern B (B1 and B4) were simultaneously found in both inpatients and outpatients. Our results indicated that PFGE with SmaI chromosomal digestion is a suitable technique to establish the inter-strain genetic relatedness of M. catarrhalis, and suggested that the outbreak of M. catarrhalis occasionally included miscellaneous PFGE patterns. The results also showed that PFGE patterns of M. catarrhalis isolates were similar between hospital- and community-acquired respiratory infections. Analysis of the subtypes suggested that there might be some association between hospital- and community-acquired respiratory infections caused by M. catarrhalis.     

Identification of elongation factor-1alpha as a Ca2+/calmodulin-binding protein in Tetrahymena cilia

Calmodulin (CaM) is known to be a ciliary component. However, the function of CaM in cilia or flagella has not been well understood. Immunoelectron microscopy using anti-CaM antibody showed that CaM was localized on the axonemal microtubules (MTs) and matrix of Tetrahymena cilia. To investigate the signal transduction of Ca(2+)/CaM in cilia, we performed Ca(2+)/CaM-affinity column chromatography in the membrane and matrix fraction. Elongation factor-1alpha (EF-1alpha) was identified as a Ca(2+)/CaM-binding protein in cilia. EF-1alpha is a highly conserved protein and functions in protein translation. In addition, EF-1alpha has been reported to interact with MTs and F-actin in several organisms. Immunoelectron microscopy showed that EF-1alpha was localized on the axonemal MTs. However, in immunoblot analysis, EF-1alpha was mainly extracted in the membrane and matrix fraction from the axonemal MTs by 1% Triton X-100 extraction. These results suggest that interaction between EF-1alpha and axonemal MTs is weak and sensitive to treatment with 1% Triton X-100 and that EF-1alpha mediates between axonemal MTs and CaM in the presence of Ca(2+). Moreover, EF-1alpha was also localized in cilia of Paramecium, suggesting that EF-1alpha functions as a target protein of Ca(2+)/CaM in ciliate cilia.     

Possible association of BLM in decreasing DNA double strand breaks during DNA replication

Bloom's syndrome (BS) is a rare genetic disorder and the cells from BS patients show genomic instability and an increased level of sister chromatid exchange (SCE). We generated BLM(-/-) and BLM(-/-)/RAD54(-/-) DT40 cells from the chicken B-lymphocyte line DT40. The BLM(-/-) DT40 cells showed higher sensitivity to methyl methanesulfonate and elevated levels of SCE as expected. The targeted integration frequency was also increased remarkably in BLM(-/-) cells. The SCE frequency increase in BLM(-/-) cells was considerably reduced and the enhanced targeted integration observed in BLM(-/-) cells was almost completely abolished in BLM(-/-)/RAD54(-/-) cells, indicating that a large portion of the SCE in BLM(-/-) cells occurs via homologous recombination, and homologous recombination events increase with the defect of BLM function. The BLM(-/-)/RAD54(-/-) cells showed a slow growth phenotype and an increased incidence of chromosome-type breaks/gaps while each single mutant showed relatively small numbers of chromosome-type breaks/gaps.     

Meiotic cytokinetic apparatus in the formation of the linear spore tetrads of Conocephalum japonicum (Bryophyta)

Most bryophytes produce tetrahedral spore tetrads. However, linear spore tetrads have been reported to occur in Conocephalum japonicum (Thunb.) Grolle. In this study, the distribution of microtubules (MTs) during meiosis in C. japonicum was examined to determine the division pattern resulting in a linear tetrad. Spore mother cells in the pre-meiotic stage were cylindrical with randomly distributed cytoplasmic MTs. In the prophase-metaphase transition, spindle MTs replaced cytoplasmic MTs and a barrel-shaped spindle with two flattened poles developed. Cortical MT arrays were not detectable throughout meiosis. Although a phragmoplast appeared between sister nuclei in telophase-I, it disappeared without expanding to the parental cell wall. Metaphase-II spindles oriented parallel to the long axis of the cell and in tandem to each other resulted in a linear arrangement of telophase nuclei. Radial arrays of MTs developed from the nuclear surfaces and three phragmoplasts appeared among the four nuclei to produce four spores. Two phragmoplasts separating the paired sister nuclei appeared prior to the appearance of a phragmoplast between non-sister nuclei. The MT cycle is basically the same as that reported in meiosis of C. conicum, which produces non-linear tetrads. A morphometric study indicated that the difference in the division pattern between C. conicum and C. japonicum is due to a difference in the shape of spore mother cells. The cylindrical shape of sporocytes of C. japonicum restricts the orientation of spindles and phragmoplasts so that the four resultant spores are arranged linearly. Received: 22 April 1998 / Accepted: 15 May 1998     

Preparation of human immortalized B-cells secreting antibodies against food allergens

An immortalized B-cell library consisting of cells secreting antibodies to food allergens has been prepared. Peripheral B-cells from seven healthy donors were transformed with Epstein-Barr virus and an immortalized human B-cell library with 2,202 ampoules of multi-clone B-lymphoblastoid cells was obtained. The B-cell library contained various types of B-lymphoblastoid cells secreting antibodies to rice, soybeans, milk, and eggs, and can be used to analyze food allergens. © Rapid Science Ltd. 1998     

Biosynthesis of branched-chain fatty acid in bacilli: FabD (malonyl-CoA:ACP transacylase) is not essential for in vitro biosynthesis of branched-chain fatty acids

It was found that the partially purified beta-ketoacyl-ACP synthase of Bacillus insolitus did not require the addition of FabD (malonyl-CoA:ACP transacylase, MAT) for the activity assay. This study therefore examined the necessity of FabD protein for in vitro branched-chain fatty acid (BCFA) biosynthesis by crude fatty acid synthetases (FAS) of Bacilli. To discover the involvement of FabD in the BCFA biosynthesis, the protein was removed from the crude FAS by immunoprecipitation. The His-tag fusion protein FabD of Bacillus subtilis was expressed in Escherichia coli and used for the preparation of antibody. The rabbit antibody raised against the expressed fusion protein specifically recognized the FabD in the crude FAS of B. subtilis. Evaluation of the efficacy of the immunoprecipitation showed that a trace of FabD protein was present in the antibody-treated crude FAS. However, this complete removal of FabD from the crude FAS did not abolish its BCFA biosynthesis, but only reduced the level to 50-60% of the control level for acyl-CoA primer and to 80% for alpha-keto-beta-methylvalerate primer. Furthermore, the FabD concentration did not necessarily correlate with the MAT specific activity in the enzyme fractions, suggesting the presence of another enzyme source of MAT activity. This study, therefore, suggests that FabD is not the sole enzyme source of MAT for in vitro BCFA biosynthesis, and implies the existence of a functional connection between fatty acid biosynthesis and another metabolic pathway.