The Regulatory Functions of the rolB and rolC Genes of Agrobacterium rhizogenes are Conserved in the Homologous Genes (Ngrol) of Nicotiana glauca in Tobacco Genetic Tumors

To compare patterns of expression between the Ngrol genes ofN. glauca and the Rirol genes of Agrobacterium rhizogenes, weperformed fluorometric and histochemical analysis of transgenicgenetic tumors on the hybrid of Nicotiana glauca x N. langsdorffü(Fl) that harbored a rß- glucuronidase (GUS) reportergene fused to the promoter of NgrolB, NgrolC, RirolB or RirolC The promoters of NgrolB and NgrolCNgrolC had 2- to 3-fold loweractivity than those of RirolB and RirolC However, the changesin patterns of GUS activity caused by deletion of NgrolB andNgrolCpromoters were similar to those of RirolB and RirolC promoters.This result suggests that the cis-acting sequences that regulatethe level of expression of RirolB and RirolC are conserved inthe NgrolB and NgrolC promoters. Furthermore, an auxin dependent(NAA-dependent) increase in GUS activity was observed in thecase of NgrolB-GUS and RirolB-GUS. Histochemical analysis showedGUS activity encoded by both NgrolB-GUS and RirolB-GUS in normal-typeFl transgenic plants was located in meristematic zones, whilethat encoded by NgrolC-GUS and RirolC-GUS was detected mainlyin vascular systems of various organs. Thus, the patterns ofexpression of the Ngrol genes were the same as those of theRirol genes in terms of promotion by auxin and tissue-specificity,indicating that regulatory mechanisms for both sets of geneshave been conserved during the evolution of the genus Nicotianaafter transfer from a progenitor of Agrobacterium to that ofNicotiana. (Received May 2, 1995; Accepted June 13, 1995)     

Metabolism of14C-labeled epibrassinolide in intact seedlings of cucumber and wheat

The metabolism of exogenously applied¹⁴C-24-epibrassinolide (¹⁴C-EBR) in seedlings of cucumber and wheat was examined. Total lipids were extracted with isopropanol and chloroform, and then partitioned with water. More than 80% of radioactivity was distributed to chloroform-phase. Concentrated chloroform-phase was applied to silica gel plate and was developed with chloroform-ethanol (5:1 v/v). Rf value of original¹⁴C-EBR was about 0.6. In cucumber leaves harvested after 2 day-culture, three peaks were detected at Rf 0.11, 0.47 and 0.84. In cucumber petioles of 2 day-culture, however, a major peak was detected at Rf 0.90. But¹⁴C-EBR was hardly metabolized in hypocotyls and roots after 2 days. In wheat leaves harvested just after pulse labeling, a peak was detected at Rf 0.63. By further analysis of this peak using ODS-HPLC, however, an original peak of¹⁴C-EBR and two metabolites having higher polarity were detected. In wheat leaves harvested after 2 day-culture, the profile of TLC scanning was similar to that just after pulse labeling, although, an original peak of¹⁴C-EBR was no longer detected by ODS-HPLC. In wheat roots,¹⁴C-EBR was hardly metabolized. These results indicate that¹⁴C-EBR occurring in leaves and petioles is metabolized to produce several kinds of metabolites.     

High-Speed Video-Oculography for Measuring Three-Dimensional Rotation Vectors of Eye Movements in Mice

Background

The mouse is the most commonly used animal model in biomedical research because of recent advances in molecular genetic techniques. Studies related to eye movement in mice are common in fields such as ophthalmology relating to vision, neuro-otology relating to the vestibulo-ocular reflex (VOR), neurology relating to the cerebellum’s role in movement, and psychology relating to attention. Recording eye movements in mice, however, is technically difficult.

Methods

We developed a new algorithm for analyzing the three-dimensional (3D) rotation vector of eye movement in mice using high-speed video-oculography (VOG). The algorithm made it possible to analyze the gain and phase of VOR using the eye’s angular velocity around the axis of eye rotation.

Results

When mice were rotated at 0.5 Hz and 2.5 Hz around the earth’s vertical axis with their heads in a 30° nose-down position, the vertical components of their left eye movements were in phase with the horizontal components. The VOR gain was 0.42 at 0.5 Hz and 0.74 at 2.5 Hz, and the phase lead of the eye movement against the turntable was 16.1° at 0.5 Hz and 4.88° at 2.5 Hz.

Conclusions

To the best of our knowledge, this is the first report of this algorithm being used to calculate a 3D rotation vector of eye movement in mice using high-speed VOG. We developed a technique for analyzing the 3D rotation vector of eye movements in mice with a high-speed infrared CCD camera. We concluded that the technique is suitable for analyzing eye movements in mice. We also include a C++ source code that can calculate the 3D rotation vectors of the eye position from two-dimensional coordinates of the pupil and the iris freckle in the image to this article.     

Structure and expression of genes coding for xylan-degrading enzymes of Bacillus pumilus

The complete nucleotide sequence of the beta-xylosidase gene (xynB) of Bacillus pumilus IPO and its flanking regions was established. A 1617-bp open reading frame for beta-xylosidase, a homodimer enzyme, was observed. The amino acid sequence of the N-terminal region and the molecular mass 62607 Da) of the beta-xylosidase subunit, deduced from the DNA sequence, agreed with the result obtained with the purified enzyme. The Shine-Dalgarno sequence was found 8 bp upstream of the initiation codon, ATG. The xylanase gene (xynA) of the same strain was 4.6 kbp downstream of the 3' end of xynB, and its DNA sequence was reported in our previous paper [Fukusaki, E., Panbangred, W., Shinmyo, A. & Okada, H. (1984) FEBS Lett. 171, 197-201]. The results of the Northern hybridization suggested that the mRNA of xynA and xynB were produced separately. The 5' and 3' ends of the xynA and xynB gene were mapped with nuclease S1. The '-10' regions for promoter sequences of both genes were similar to the consensus sequence for B. subtilis RNA polymerases, the '-35' regions were different from all the known promoters for B. subtilis RNA polymerases.     

Vascular endothelial growth factor in patients with high-altitude pulmonary edema.

To examine the role of VEGF in the pathogenesis of high-altitude pulmonary edema (HAPE), we measured the concentrations of VEGF in venous serum and bronchoalveolar lavage fluid in patients with HAPE and in healthy volunteers. The VEGF in venous serum of the patients was normal at admission and significantly increased at recovery. Similarly, the VEGF in bronchoalveolar lavage fluid of the patients was increased at recovery compared with admission, but values at both admission and recovery were significantly lower than those of the controls. The present finding suggests that VEGF probably is destroyed in the lung of HAPE, and it appears less likely to have a critical part in the pathogenesis of HAPE but has rather an important role in the repair process for the impaired cell layer.     

The development of colitogenic CD4(+) T cells is regulated by IL-7 in collaboration with NK cell function in a murine model of colitis

We previously reported that IL-7(-/-)RAG(-/-) mice receiving naive T cells failed to induce colitis. Such abrogation of colitis may be associated with not only incomplete T cell maintenance due to the lack of IL-7, but also with the induction of colitogenic CD4(+) T cell apoptosis at an early stage of colitis development. Moreover, NK cells may be associated with the suppression of pathogenic T cells in vivo, and they may induce apoptosis of CD4(+) T cells. To further investigate these roles of NK cells, RAG(-/-) and IL-7(-/-)RAG(-/-) mice that had received naive T cells were depleted of NK cells using anti-asialo GM1 and anti-NK1.1 Abs. NK cell depletion at an early stage, but not at a later stage during colitogenic effector memory T cell (T(EM)) development, resulted in exacerbated colitis in recipient mice even in the absence of IL-7. Increased CD44(+)CD62L(-) T(EM) and unique CD44(-)CD62L(-) T cell subsets were observed in the T cell-reconstituted RAG(-/-) recipients when NK cells were depleted, although Fas, DR5, and IL-7R expressions in this subset differed from those in the CD44(+)CD62L(-) T(EM) subset. NK cell characteristics were the same in the presence or absence of IL-7 in vitro and in vivo. These results suggest that NK cells suppress colitis severity in T cell-reconstituted RAG(-/-) and IL-7(-/-)RAG(-/-) recipient mice through targeting of colitogenic CD4(+)CD44(+)CD62L(-) T(EM) and, possibly, of the newly observed CD4(+)CD44(-)CD62L(-) subset present at the early stage of T cell development.     

Prostaglandin E₂ increases both osteoblastic and osteoclastic activity in the scales and participates in calcium metabolism in goldfish

Using our original in vitro assay system with goldfish scales, we examined the direct effect of prostaglandin E? (PGE?) on osteoclasts and osteoblasts in teleosts. In this assay system, we measured the activity of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) as respective indicators of each activity in osteoblasts and osteoclasts. ALP activity in scales significantly increased following treatment at high concentration of PGE?(10?? and 10?? M) over 6 hrs of incubation. At 18 hrs of incubation, ALP activity also significantly increased in the PGE? (10?? to 10?? M)-treated scale. In the case of osteoclasts, TRAP activity tended to increase at 6 hrs of incubation, and then significantly increased at 18 hrs of incubation by PGE? (10(-7) to 10?? M) treatment. At 18 hrs of incubation, the mRNA expression of osteoclastic markers (TRAP and cathepsin K) and receptor activator of the NF-κB ligand (RANKL), an activating factor of osteoclasts expressed in osteoblasts, increased in PGE? treated-scales. Thus, PGE? acts on osteoblasts, and then increases the osteoclastic activity in the scales of goldfish as it does in the bone of mammals. In an in vivo experiment, plasma calcium levels and scale TRAP and ALP activities in the PGE?-injencted goldfish increased significantly. We conclude that, in teleosts, PGE? activates both osteoblasts and osteoclasts and participates in calcium metabolism.     

Neuroglycan C is a novel midkine receptor involved in process elongation of oligodendroglial precursor-like cells

Midkine is a heparin-binding growth factor that promotes cell attachment and process extension in undifferentiated bipolar CG-4 cells, an oligodendroglial precursor cell line. We found that CG-4 cells expressed a non-proteoglycan form of neuroglycan C, known as a part-time transmembrane proteoglycan. We demonstrated that neuroglycan C before or after chondroitinase ABC treatment bound to a midkine affinity column. Neuroglycan C lacking chondroitin sulfate chains was eluted with 0.5 m NaCl as a major fraction from the column. We confirmed that CG-4 cells expressed two isoforms of neuroglycan C, I, and III, by isolating cDNA. Among three functional domains of the extracellular part of neuroglycan C, the chondroitin sulfate attachment domain and acidic amino acid cluster box domain showed affinity for midkine, but the epidermal growth factor domain did not. Furthermore, cell surface neuroglycan C could be cross-linked with soluble midkine. Process extension on midkine-coated dishes was inhibited by either a monoclonal anti-neuroglycan C antibody C1 or a glutathione S-transferase-neuroglycan C fusion protein. Finally, stable transfectants of B104 neuroblastoma cells overexpressing neuroglycan C-I or neuroglycan C-III attached to the midkine substrate, spread well, and gave rise to cytoskeletal changes. Based on these results, we conclude that neuroglycan C is a novel component of midkine receptors involved in process elongation.