GATAe-dependent and -independent expressions of genes in the differentiated endodermal midgut of Drosophila

Two sequentially-expressed GATA factor genes, serpent (srp) and GATAe, are essential for development of the Drosophila endoderm. The earliest endodermal GATA gene, srp, has been thought to specify the endodermal fate, activating the second GATA gene GATAe, and the latter continues to be expressed in the endodermal midgut throughout life. Previously, we proposed that GATAe establishes and maintains the state of terminal differentiation of the midgut, since some functional genes in the midgut require GATAe activity for their expression. To obtain further evidence of the role of GATAe, we searched for additional genes that are expressed specifically in the midgut in late stages, and examined responses of a total of selected 15 genes to the depletion and overexpression of GATAe. Ten of the 15 genes failed to be expressed in the embryo deficient for GATAe activity, but, the other five genes did not require GATAe. Instead, srp is required for activating the five genes. These observations indicate that GATAe activates a major subset of genes in the midgut, and some other pathway(s) downstream of srp activates other genes.     

A functionalized 20-residue peptaibol derivative for nucleic acid delivery

Based on the membrane-modifying peptaibol trichocellin-A-I (1) from Trichoderma viride, we designed a vehicle for the cellular delivery of antisense oligodeoxynucleotides by attaching a (Lys)10 stretch to the C-terminus of 1. The resulting transporter peptide 2, prepared by solid-phase synthesis using Fmoc protocol in combination with amino acid fluorides, was found to be mainly alpha-helical in solution, in contrast to its precursors 1 and 3. The uptake of the complex formed between carrier 2 and a fluorescence-tagged oligonucleotide, i.e., 4, was studied at different charge ratios by confocal laser-scanning microscopy, using two different eukaryotic cell lines: mouse embryonal fibroblast (NIH3T3) and human lung carcinoma (A549) cells. Peptide 2 readily translocated 4 into the cytoplasms of NIH3T3 cells. However, the peptide/oligonucleotide complex was accumulated around the plasma membrane of the A549 cells.     

Identification of acetic acid bacteria isolated from Indonesian sources,especially of isolates classified in the genus Gluconobacter

Sixty-four strains of acetic acid bacteria were isolated from Indonesian sources such as fruits, flowers, and fermented foods by the enrichment culture at pH 3.5. Forty-five strains were routinely identified as Acetobacter strains because of their oxidation of acetate and lactate to carbon dioxide and water and their Q-9 isoprenolog, corresponding to 70% of all the 64 acetic acid bacteria isolated. Eight isolates were identified as Gluconacetobacter strains because of their oxidation of acetate and lactate and their Q-10 isoprenolog, occupying 13% of all the isolates. The remaining 11 isolates, accommodated in the genus Gluconobacter because of no oxidation of acetate and lactate and because of their Q-10 isoprenolog, accounted for 17% of all the isolates. They were divided into two groups based on DNA base compositions. One comprised the seven isolates, which had high G1C contents of DNA ranging from 60.3 to 63.5 mol% and of which DNAs hybridized with that of the type strain of Gluconobacter oxydans at values of 64-94% of DNA relatedness. The other comprised the remaining four isolates, which had low G+C contents of DNA ranging from 57.5 to 57.7 mol% and of which DNAs hybridized with that of the type strain of Gluconobacter frateurii at values of 63-77% of DNA relatedness. The high values of DNA relatedness, 84 to 96%, were obtained between the type strains of Gluconobacter cerinus and Gluconobacter asaii.     

Zebrafish serotonin-N-acetyltransferase-2 gene regulation: pineal-restrictive downstream module contains a functional E-box and three photoreceptor conserved elements

Pineal function is defined by a set of very narrowly expressed genes that encode proteins required for photoperiodic transduction and rhythmic melatonin secretion. One of these proteins is serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT), which controls the daily rhythm in melatonin production. Here, pineal-specific expression of the zebrafish aanat-2 (zfaanat-2) was studied using in vivo transient expression analyses of promoter-reporter constructs; this revealed that specificity is determined by two regions located 12 kb away from each other. One is the 5'-flanking region, and the other is a 257-bp sequence, located 6 kb downstream of the transcribed region. This 3'-sequence, designated pineal-restrictive downstream module (PRDM), has a dual function: enhancement of pineal expression and inhibition of extrapineal expression. The former is an autonomic property of PRDM whereas the later function requires interaction with the upstream regulatory region of zfaanat-2. Functional analyses of the PRDM sequence revealed that three photoreceptor conserved elements (TAATC) and a single perfect E-box (CACGTG) are crucial for the dual function of PRDM. These results indicate that pineal specificity of zfaanat-2 is determined by the dual functionality of the PRDM and the interaction between upstream regulatory region and downstream photoreceptor conserved elements and E-box element.     

Six1 controls patterning of the mouse otic vesicle

Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wild-type otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1- and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.     

Evaluation of a high-affinity QCM immunosensor using antibody fragmentation and 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer

This study evaluated construction of a highly affinitive quartz crystal microbalance (QCM) immunosensor using anti-C-reactive protein (CRP) antibody and its fragments for CRP detection. Three types of antibody were immobilized on the surface of a QCM via covalent-bounding. Then affinity was evaluated through antigen-antibody binding between CRP and its antibody. Affinity between antigen-antibody was shown to be highest when anti-CRP F(ab')2-IgG antibody (70 microg/mL) was immobilized on the QCM. In case of anti-CRP F(ab')2-IgG antibody, affinity which was attributable to antigen-antibody binding was almost twice that of anti-CRP IgG antibody, which is used conventionally for QCM immunosensors. In addition, when it was treated with 2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate, so-called MPC polymer, highly affinitive and selective immunosensing for CRP was achieved without non-specific binding from plasma proteins in human serum. When anti-CRP F(ab')2-IgG antibody was immobilized on the QCM, the detection limit and the linearity of CRP calibration curve were achieved at concentrations from 0.001 to 100 microg/dL even during investigation in serum samples. Experimental results verified the successful construction of a highly affinitive and selective QCM-immunosensor which was modified with anti-CRP F(ab')2-IgG antibody and MPC polymer.