Kenichi P. Takahashi

Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome

Generation of RNA dimeric form of the human immunodeficiency virus type 1 (HIV-1) genome is crucial for viral replication. The dimerization initiation site (DIS) has been identified as a primary sequence that can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem. It has been reported that HIV-1 RNA fragments containing the DIS form two types of dimers, loose dimers and tight dimers. The loose dimers are spontaneously generated at the physiological temperature and converted into tight dimers by the addition of nucleocapsid protein NCp7. To know the biochemical process in this two-step dimerization reaction, we chemically synthesized a 39-mer RNA covering the entire DIS sequence and also a 23-mer RNA covering the self-complementary loop and its flanking stem within the DIS. Electrophoretic dimerization assays demonstrated that the 39-mer RNA reproduced the two-step dimerization process, whereas the 23-mer RNA immediately formed the tight dimer. Furthermore, deletion of the bulge from the 39-mer RNA prevented the NCp7-assisted tight-dimer formation. Therefore, the whole DIS sequence is necessary and sufficient for the two-step dimerization. Our data suggested that the bulge region regulates the stability of the stem and guides the DIS to the two-step dimerization process.

S1-1 nuclear domains: characterization and dynamics as a function of transcriptional activity

Background information. The RNA‐binding protein S1‐1, also called RBM10 (RNA‐binding motif 10), is a paralogue of putative tumour suppressor RBM5 and has been correlated with cancer proliferation and apoptosis. In the present study, we have investigated the cell biology of S1‐1.

The occurrence of IgG in the endolymphatic sac of the guinea pig

SummaryWe investigated the occurrence of immunoglobulin G (IgG) in the endolymphatic sac (ES) of the guinea pig in order to show that the ES is an organ involved in the immunoreactivity of the inner ear. By using an immunohistological technique, we were able to show that IgG is located mainly in the subepithelial layer of the ES. We also found that IgG is present in some epithelial cells of the ES as well as in free floating cells in the ES lumen. Although plasma cells have been described previously in the subepithelial layer, none could be recognized in this layer of the ES in our specimens. Our results suggest that the ES is an immunoreactive organ of the inner ear in some pathological states, while IgG in the normal ES is primarily of systemic origin.

Association of hnRNP S1 proteins with vimentin intermediate filaments in migrating cells

S1 proteins C2 and D2 are multifunctional hnRNP proteins acting as transcriptional regulators in the nucleus. Immunofluorescence staining of various cells in culture revealed that S1 proteins also occur in the cytoplasm, often in association with vimentin intermediate filaments (VFs). Here, we verified the association of S1 proteins with vimentin using vimentin-deficient cells, crosslinking and immunoprecipitation, and further investigated the biological significance of this association. S1 proteins on VFs, referred to here as S1 fibers, were lost in highly confluent cells, where cell proliferation and cellular metabolic activity greatly decreased owing to cell density-dependent arrest. However, the disappearance of S1 fibers was not related to these reduced activities, but to inhibited cell migration. Although undetected in cells of non-migratory tissues as well as in confluent cultured cells, S1 fibers were found in all migratory cells examined, such as cultured cells in scratch/wound experiments, blood neutrophils and monocytes, and fibroblasts engaging in tissue healing. In addition, S1 fibers reappeared even in confluent cells when VFs were induced to reorganize with okadaic acid. We propose that S1 proteins occur in association with VFs in migratory cells. Possible participation of S1 proteins in the formation/reorganization of VFs is discussed.

Immunohistochemical demonstration of nuclear S1 proteins in various cells

SummaryS1 proteins are present in the nuclear structures sensitive to DNases and RNase. To examine localization of these proteins, an antibody was raised in a rabbit. Indirect immunofluorescence staining revealed that S1 proteins located in the extranucleolar nuclear regions of quiescent myocardial and cerebellar cells as well as actively duplicating mouse 3T3 fibroblasts. They located in euchromatin regions of thymus lymphocytes, with a characteristic aster-like immunofluorescence pattern, and on the border of condensed chromatin areas by deposition of immunogold particles in ultrathin sections of thymus. Thus, S1 proteins may be in a nuclear function assigned to the border of heterochromatin areas, and other than synthesis of DNA or of ribosomal RNA. Possible involvement of S1 proteins in the extranucleolar RNA synthesis is discussed.

Natural Killer Cell Response in the Inner Ear

Heterologous tumor cells (human chronic myelogenous leukemia K-562 cells) were injected into the perilymph and skin in guinea pigs in order to study the induction of NK cell activity in the inner ear. An in vitro transmission electron microscopic and immunohistochemical study showed that K-562 cells were attacked by guinea pig large granular lymphocytes. K-562 cells injected through the round window membrane were found to be targeted by NK cells emerging from surrounding venules after 7 to 9 days. During this time morhological changes occurred in the organ of Corti and stria vacularis. These findings suggest that the inner ear response to foreign cells induces activation and invasion of NK cells which occur relatively late compared with those in other organs such as skin.

Localization of terminal deoxynucleotidyl transferase (TdT) in rat thymus using immunoperoxidase methods

SummaryIndirect and peroxidase anti-peroxidase (PAP) immunoenzymatic methods were used to detect terminal deoxynucleotidyl transferase (TdT) in imprints and formalin-fixed paraffin sections of normal rat thymus. TdT is found in the nuclei of small lymphocytes in imprint samples from neonatal and adult rat thymus, showing granular or circular patterns of peroxidase reaction products. Diffuse brown reaction products of peroxidase are located in both the nuclei and cytoplasm of medium and large lymphocytes. Indirect measurements show that, as age progresses, the percentage of peroxidase-positive cells decreases in all types of lymphocytes, from 72.4% on the 11th day to 54.8% in the 5th month, whereas that of negative cells increases from 14.4% to 39.4%. In formalin-fixed paraffin sections, peroxidase-positive lymphocytes are found mainly in the cortex and cortico-medullary boundary, and only rarely in the medulla.

Quick gas chromatographic method for measuring the water content of brain tissue: with reference to age-related changes of rat neocortex.

A quick and accurate method for estimating tissue water content by gas chromatography was developed. Age-related changes in tissue water content in the neocortex of rat brain were easily determined by this method.