Effect of NMDA receptor antagonist on proliferation of neurospheres from embryonic brain

The N-methyl-D-aspartate (NMDA) receptor, a subtype of ionotropic glutamate receptors, plays an important role in the regulation of neuronal development, learning and memory, and neurodegenerative diseases. NMDA receptor blockade enhances neurogenesis in the hippocampal dentate gyrus in vivo. The effect of NMDA receptor antagonist on proliferation of neural progenitor cells, however, remains to be determined. We investigated changes in the diameter and number of neurospheres derived from the embryonic rat brain after NMDA receptor blockade. Cortical progenitor cells were isolated from gestational day 18 fetal rats according to the Percoll density gradient method. Cultured spheres expressed neural progenitor markers, musashi-1 and nestin. Immunohistochemical analysis demonstrated that cells in Dulbecco's modified Eagle medium/F12 containing 1% fetal bovine serum on day 8 differentiated to MAP-2-positive neurons and GFAP-positive astrocytes. The expression of NR1 and NR2B subunits of the NMDA receptor in neurospheres was detected. Neither brief nor sustained exposure to NMDA altered the diameter and number of neurospheres. Brief exposure to 30 μM MK-801, an NMDA receptor antagonist, decreased the diameter of neurospheres. Sustained exposure to 30 μM MK-801 decreased the diameter and number of neurospheres. Our results provide evidence that MK-801 directly decreased proliferation of neural progenitor cells.     

Vertical transmission of human T-cell leukemia virus type I (HTLV-I): Detection of proviral DNA in HTLV-I carrier gravida

The seroprevalence rate of human T-cell leukemia virus type I (HTLV-I) in pregnant women in the Osaka district was determined by enzyme-linked immunosorbent assay and Western blot analysis. Twenty-one (1.0%) of 2192 samples tested were positive for both assays and the seropositive parturients were found to be integrated with HTLV-I proviral DNA in their mononuclear cells by a DNA dot blot hybridization assay using HTLV-I DNA probe or by a selective DNA amplification technique using the polymerase chain reaction (PCR). On the other hand, proviral DNA was not detected in cord blood of the neonates born to the carrier mothers, indicating that transplacental infection of HTLV-I during pregnancy could be excluded. The results support the hypothesis that postpartum infection via breast milk plays a significant role among the possible perinatal transmission routes.     

Unique three-way translocation, t(3;14;18)(q27;q32;q21), in follicular lymphoma

A 43-year-old woman was diagnosed as having stage IV follicular lymphoma. Phenotypically, the lymphoma cells were CD5(-), CD10(+), CD19(+), CD20(+), CD23(-), HLA-DR(+), and IgM-lambda(+). Conventional chromosomal analysis showed a three-way t(3;14;18)(q27;q32;q21) in the lymphoma cells, which was confirmed by spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH). Immunohistochemistry revealed that both BCL2 and BCL6 proteins were expressed in the lymphoma cells, whereas only the BCL6 gene, and not the BCL2 gene, was rearranged by Southern blotting. The patient received combination chemotherapy and has been well for 3 years. This is the first reported case showing a three-way translocation involving 2 major lymphoma-specific abnormalities, 3q27 and t(14;18)(q32;q21).     

Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia

Brain ischemia induces a marked response of resident microglia and hematopoietic cells including monocytes/macrophages. The present study was designed to assess the distribution of microglia/macrophages in cerebral ischemia using bone marrow chimera mice known to express enhanced green fluorescent protein (EGFP). At 24 h after middle cerebral artery occlusion (MCAO), many round-shaped EGFP-positive cells migrated to the ischemic core and peri-infarct area. At 48-72 h after MCAO, irregular round- or oval-shaped EGFP/ionized calcium-binding adapter molecule 1 (Iba 1)-positive cells increased in the transition zone, while many amoeboid-shaped or large-cell-body EGFP/Iba 1-positive cells were increased in number in the innermost area of ischemia. At 7 days after MCAO, many process-bearing ramified shaped EGFP/Iba 1-positive cells were detected in the transition to the peri-infarct area, while phagocytic cells were distributed in the transition to the core area of the infarction. The distribution of these morphologically variable EGFP/Iba 1-positive cells was similar up to 14 days from MCAO. The present study directly showed the migration and distribution of bone marrow-derived monocytes/macrophages and the relationship between resident microglia and infiltrated hematogenous element in ischemic mouse brain. It is important to study the distribution of intrinsic and extrinsic microglia/macrophage in ischemic brain, since such findings may allow the design of appropriate gene-delivery system using exogenous microglia/macrophages to the ischemic brain area.     

Phylogenetic Classification of Trichophyton mentagrophytes Complex Strains Based on DNA Sequences of Nuclear Ribosomal Internal Transcribed Spacer 1 Regions

Using internal transcribed spacer 1 (ITS1) region ribosomal DNA sequences from 37 stock strains and clinical isolates provisionally termed Trichophyton mentagrophytes complex in Japan, we demonstrated the mutual phylogenetic relationships of these strains. Members of this complex were classified into 3 ITS1-homologous groups and 13 ITS1-identical groups by their sequences. ITS1-homologous group I consists of Arthroderma vanbreuseghemii, T. mentagrophytes human isolates, and several strains of T. mentagrophytes animal isolates. Five strains of Arthroderma simii form a cluster comprising ITS1-homologous group II. The Americano-European and African races of Arthroderma benhamiae, T. mentagrophytes var. erinacei, and one strain of a T. mentagrophytes animal isolate constitute ITS1-homologous group III. According to the phylogenetic tree constructed with Trichophyton rubrum as an outgroup, ITS1-homologous groups I and II comprised a monophyletic cluster and ITS1-homologous group III constituted another cluster which was rather distant from the others in the complex. This system was applicable to the phylogenetic analysis of closely related strains. Using this technique, human and animal isolates of T. mentagrophytes were also clearly distinguishable from each other.Dermatophytes have the capacity to invade keratinized tissues, that is, the skin, hair, and nails, of humans and other animals to produce an infection, dermatophytosis, referred to as ringworm or tinea. Trichophyton mentagrophytes (8) is known as a complex species (22) and is one of the major pathogens causing this infection (23). Using mating tests and microscopic observation of ascospores, three perfect fungal states of T. mentagrophytes have been identified in this imperfect or conidial “species.” They are Arthroderma vanbreuseghemii, Arthroderma simii, and Arthroderma benhamiae (1, 20, 22), the latter being classified into two races, American-European and African (21). The phylogeny of T. mentagrophytes, however, remains unclear because the phenotypic features of members of the T. mentagrophytes complex are poor and many isolates from medical and veterinary samples have lost their sexual activity (22). From a clinical point of view, because the T. mentagrophytes complex includes both anthrophilic and zoophilic species (23), it is important to have a reliable method of identifying the human-pathogenic species of the complex. Establishment of the phylogenetic classification of this complex has been achieved by molecular biological studies on the phylogeny of pathogenic fungi, primarily using the G+C content of chromosomal DNA (5), total DNA homology (6), restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA) (7, 13, 17, 18), and the base sequence of the 18S (11) or 28S (14) rRNA or rRNA gene (rDNA). However, for dermatophytes, including T. mentagrophytes, the phylogenic relationship or species-specific sequences cannot be defined by these methods, because the members of this group of fungi are phylogenetically and taxonomically very closely related. Specific DNA sequences of the internal transcribed spacer 1 region (ITS1) of the rDNA in the T. mentagrophytes complex, mainly of strains stocked in Japan, were therefore determined and phylogenetically analyzed. ITS1 is located between the 18S and 5.8S rDNAs. As reported previously, the variable ITS regions have proven useful in resolving relationships between close taxonomic relatives (2, 3, 15). We were able to successfully differentiate between members of the T. mentagrophytes complex and a related species, Trichophyton rubrum, and to demonstrate their phylogenetic relationship by base pair comparisons of ITS1 regions.     

Endocytosis of human thyroglobulin (TG) in adenomatous goiter studied by an immuno-electron microscopic procedure

Five cases of adenomatous goiter have been studied by an electron microscope using an immuno-reaction for thyroglobulin (TG) and focusing on the mechanism of endocytosis. Positive stain for TG was demonstrated in follicular lumina, large reabsorbed colloid droplets and small subapical vesicles. Endocytotic vesicles ranging from 320 nm to 1600 nm in diameter were observed in the cytoplasm as pits in the apical plasma membrane. Some of them showed direct connection with the positive stain for TG in the follicular lumen and the others were completely ingested in the cytoplasm. With statistic analysis, a majority of the vesicles showing the positive stain for TG in the cytoplasm distributed in the range of 200 nm to 1200 nm in diameter with the peak in 300 nm to 399 nm and was situated within an extent of the diameter measured from the endocytotic vesicles. Engulfment of colloid by pseudopods and fusion of the reabsorbed colloid droplets were encountered as extremely rare findings and appeared to play no major role for formation of large colloid droplets in adenomatous goiter.     

HLA antigens in Behçet''s disease with refractory ocular attacks

HLA class I, II, and III antigens were studied in Japanese patients with Beh?et's disease with refractory ocular attacks. In addition to the increased frequency of B51, DQw3, especially TA10-negative DQw3, was increased and DQw1 was decreased significantly in this subgroup of Beh?et's disease. As for complement markers, C4A Q0 was increased. A rare variant of BF S07 was first observed in Japanese. Although the mechanism for the DQw3 association is obscure, a possible hypothesis is that an immune-response or immune-suppression gene linked to the DQ antigens modulates the disease severity and the efficacy of treatments.     

Further development of a recombinant feline herpesvirus type 1 expressing the Gag protein of feline immunodeficiency virus

Summary.  We have previously reported the construction of a recombinant feline herpesvirus type 1 (FHV-1), designated C7301ddlTK-gag, expressing the Gag precursor protein of feline immunodeficiency virus (FIV). In this study, we report the construction of a further recombinant FHV-1 (ddlTK(gBp)-gag) which carries an FHV-1 gB promoter sequence upstream of the FIV gag gene of C7301ddlTK-gag. Strong expression of the FIV Gag protein by ddlTK(gBp)-gag was confirmed by immunoblot analyses and enzyme-linked immunosorbent assays. Although C7301ddlTK-gag and ddlTK(gBp)-gag failed to induce anti-FIV Gag antibodies in cats, we confirmed the infectivity and stability of these recombinants in cats. Received January 14, 2000 Accepted August 4, 2000     

Simultaneous measurements of temperature and pH in vivo using NMR in conjunction with TmDOTP5-

NMR techniques for temperature and pH measurements have attracted increasing interest in recent years, motivated in part by the growing importance of medical hyperthermia for the treatment of cancer. The chemical shifts of thulium 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetrakis(methylene phosphonate) (TmDOTP5-) have been studied as a function of temperature and pH. The results demonstrate that TmDOTP5- resonance shifts are highly sensitive to temperature (approximately 1.0 ppm/degrees C) and pH (approximately 3.2 ppm/pH unit) at clinically relevant field strengths. A new method is presented which utilizes two magnetically non-equivalent protons in TmDOTP5- for simultaneous NMR measurements of both temperature and pH. The difference in the chemical shift values of pairs of 1H resonances provides a temperature sensitivity of about 1.6 ppm/ degrees C. The technique is demonstrated in live rats undergoing ultrasound-induced hyperthermia therapy.