De novo methylation of MMLV provirus in embryonic stem cells: CpG versus non-CpG methylation

Nuclear factor Y (NF-Y) is a highly conserved trimeric activator that recognizes with high specificity and affinity the widespread CCAAT box promoter element. We previously cloned the genes of 23 NF-Y genes of Arabidopsis thaliana (Gene 264 (2001) 173). Now that the Arabidopsis genome sequencing project is complete, we present the cloning, alignments and expression profiles of the remaining six genes coding for the three NF-Y subunits. Consistent with our previous reports, most of the new members of the three subunits show a unique tissue-specific pattern, while another AtNF-YC9 is rather ubiquitous.     

rugose (rg), a Drosophila A kinase anchor protein,is required for retinal pattern formation and interacts genetically with multiple signaling pathways

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the EGFR- and Notch-mediated signaling pathways. Our results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the EGFR and Notch signaling pathways.     

Direct, Ca2+-dependent interaction between tubulin and synaptotagmin I: a possible mechanism for attaching synaptic vesicles to microtubules

The synaptic vesicle protein synaptotagmin I probably plays important roles in the synaptic vesicle cycle. However, the mechanisms of its action remain unclear. In this study, we have searched for cytoplasmic proteins that interact with synaptotagmin I. We found that the cytoskeletal protein tubulin directly and stoichiometrically bound to recombinant synaptotagmin I. The binding depended on mm Ca(2+), and 1 mol of tubulin dimer bound 2 mol of synaptotagmin I with half-maximal binding at 6.6 microm tubulin. The Ca(2+) dependence mainly resulted from Ca(2+) binding to the Ca(2+) ligands of synaptotagmin I. The C-terminal region of beta-tubulin and both C2 domains of synaptotagmin I were involved in the binding. The YVK motif in the C2 domains of synaptotagmin I was essential for tubulin binding. Tubulin and synaptotagmin I were co-precipitated from the synaptosome extract with monoclonal antibodies to tubulin and SNAP-25 (synaptosome-associated protein of 25 kDa), indicating the presence of tubulin/synaptotagmin I complex and tubulin binding to synaptotagmin I in SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. Synaptotagmin I promoted tubulin polymerization and bundled microtubules in the presence of Ca(2+). These results suggest that direct interaction between synaptotagmin I and tubulin provides a mechanism for attaching synaptic vesicles to microtubules in high Ca(2+) concentrations.     

Electrical perception of "death message" in chara: analysis of rapid component and ionic process

Electrical response upon wounding was analyzed in Chara corallina. A specimen comprising two adjoining internodal cells was prepared. One cell (victim cell) was killed by cutting and any changes in the membrane potential of the neighboring cell (the receptor cell) were measured. Upon cutting the victim cell, the receptor cell generated four kinds of depolarizations: (1) rapid component, (2) slow and long-lasting component, (3) action potential and (4) small spike. Rapid and slow components were observed in most cells. On the other hand, the action potential and small spike were not always ubiquitous among specimens. When an action potential was generated just after cutting the victim cell, the rapid component could not be observed due to masking by the action potential. It was suggested that both rapid and slow components were generated at the nodal end. On the other hand, action potentials were thought to be generated at the flank of the receptor cell. High turgor pressure of the cell was necessary for generating both rapid and slow components. Experiments under K(+)-induced depolarization unequivocally showed that the Cl(-) channel at the nodal end of the receptor cell was activated upon cutting the victim cell.     

Rhizoid differentiation in Spirogyra: position sensing by terminal cells

Some species of Spirogyra anchor themselves to the substrate by differentiating rhizoids. A rhizoid is differentiated only from the terminal cell, suggesting that this cell can recognize its terminal position in a filament. In the present study, we have analyzed the mechanism for position sensing by the terminal cell. When a filament is cut, a new cell occupies the terminal position, and three phenomena are induced: (1) the cell wall of the cut cell detaches from the new terminal cell; (2) adhesive material is secreted by the terminal cell; and (3) the terminal cell begins to differentiate a rhizoid via tip growth. All of these phenomena were inhibited by adding sorbitol to the external medium, suggesting that turgor pressure is involved in position sensing by the terminal cell. The inhibition by sorbitol was reversible. Upon cutting a filament, the distal end of a new terminal cell became convex. However, when a filament was cut in the presence of sorbitol, the distal end of a new terminal cell became less convex. Either treatment with Gd(3+) or decrease in extracellular Ca(2+) resulted in inhibition of all these phenomena, suggesting possible involvement of stretch-activated ion channel in position sensing by terminal cells.     

Med,a cell-surface localized protein regulating a competence transcription factor gene,comK, in Bacillus subtilis

Med was found as a positive regulator for comK, a master regulator for late competence genes. It was found by Western analysis that the ComK level was decreased in a med mutant. Experiments using an alkaline phosphatase fusion with Med and Western analysis of Med were done because a putative lipo-modification signal is found at the N-terminus of Med. The results obtained are consistent with the localization of Med at the cell surface. An implication of the cell-surface localization of Med is discussed in terms of comK regulation.     

Apoptosis induction by wheat-flour sphingoid bases in DLD-1 human colon cancer cells

The apoptotic effects of plant sphingoid bases prepared from wheat-flour cerebroside on human colorectal cancer DLD-1 cells were examined. The viability of DLD-1 cells treated with such plant sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. Morphological changes such as condensed chromatin fragments were found, so those sphingoid bases reduced cell viability through causing apoptosis in these cells.     

Histochemical analysis of macrophage migration inhibitory factor in psoriasis vulgaris

Psoriasis is a persistent cutaneous disease characterized by skin inflammation and infiltration of immunocytes such as lymphocytes and monocytes/macrophages, concomitant with abnormal epidermal hyperproliferation. We previously showed that the serum level of macrophage migration inhibitory factor (MIF) and its production by peripheral blood mononuclear cells of patients with psoriasis were closely correlated with the severity of clinical symptoms; however, the precise role of MIF in psoriatic epidermis remains to be clarified. The current study was carried out to elucidate the possible involvement of MIF in psoriasis, using immunohistochemistry and in situ hybridization. In contrast to elevated serum MIF in psoriasis, MIF-positive staining in the lesional psoriatic epidermis was significantly decreased, as demonstrated by immunohistochemical analysis using an anti-MIF antibody. Consistent with this finding, we found, by in situ hybridization, that MIF mRNA concomitantly decreased in the psoriatic lesions. Although the reason for the different MIF levels in the psoriatic epidermis and in the circulation remains unknown, it is hypothesized that MIF, a potential growth factor, might be decreased in psoriatic lesions to counterregulate the abnormal epidermal proliferation caused by dysregulation of cytokines and growth factors.