Fumiko Maeda

Changing pitch induced visual motion illusion.

We often associate moving objects and changing pitch, e.g., falling stones with descending, and launching rockets with ascending pitch, even when these sounds do not happen in the real-world. The reason for this is unknown. Here we report an illusion in which auditory stimuli with no apparent spatial and motion information [[1–3]] alter human visual motion perception. Subjects made a two alternative forced choice (upward (Vup) or downward (Vdown) visual motion perception) while presented with two superimposed, oppositely moving gratings (experiment 1), accompanied by either an ascending or a descending pitch of pure tone, or broad-band noise (Figure 1A). Gratings with ambiguous motion accompanied by ascending pitch were more likely to be perceived as an upward motion, those accompanied by descending pitch as a downward motion, whereas noise caused no directional bias.

Transcranial magnetic stimulation: studying motor neurophysiology of psychiatric disorders

AbstractRationale. Transcranial magnetic stimulation (TMS) is a noninvasive tool that directly stimulates cortical neurons by inducing magnetic and secondary electric fields. Traditionally TMS has been used to study the motor neurophysiology of healthy subjects and those with neurological disorders.Objective. Given the known motor dysfunctions in many psychiatric disorders supplemental usage of TMS to study the underlying pathophysiology of certain psychiatric disorders and to assess treatment outcomes is underway. Such studies include examination of motor neuronal membrane, corticospinal and intracortical excitability. Our objective is to overview the past findings.Methods. We review the past literature that used TMS as an assessment tool in psychiatric disorders such as schizophrenia, mood disorders, Tourettes syndrome, obsessive-compulsive disorder, attention-deficit hyperactivity disorder, and substance abuse.Results. While the findings are still preliminary due to small sample-size, inconsistent patient population (diagnosis, medication), differences in methodology between research groups, studies restricted to the motor region and possible lack of sensitivity and specificity, the studies are yielding interesting results which could potentially lead to trait- and state-markers of psychiatric disorders.Conclusions. Future studies using TMS alone or in combination with other neuroimaging techniques promise to further expand the application of TMS from studies of motor excitability to higher cognitive functions.

Carbohydrate-dependent signaling from the phosphatidylglucoside-based microdomain induces granulocytic differentiation of HL60 cells

Glycosphingolipids form glycosphingolipid signaling microdomains. Here, we report an unrecognized type of phosphatidylglucoside (PhGlc)-based lipid microdomain in HL60 cells. Treatment of cells with rGL-7, which preferentially reacts with PhGlc, induced differentiation of HL60 cells. This was manifested by the appearance of nitroblue tetrazolium-positive cells together with CD38 expression and c-Myc down-regulation. We determined the molecular mechanisms underlying early stages of signal transduction. rGL-7 treatment induced rapid tyrosine phosphorylation of Src family protein kinases Lyn and Hck. Reduction of endogenous cholesterol after application of methyl-β-cyclodextrin suppressed rGL-7-stimulated tyrosine phosphorylation. Phosphorylated proteins and PhGlc colocalized in the Triton X-100 insoluble, light buoyant density fraction after sucrose gradient ultracentrifugation of HL60 cell lysates. This suggests PhGlc-based microdomain is involved in GL-7 signaling. Ligation of known components of microdomains, such as sphingomyelin and ganglioside GM1, with corresponding antibodies failed to induce differentiation and tyrosine phosphorylation. These results show that PhGlc constitutes a previously undescribed lipid signaling domain, and the glucose residue of PhGlc is critical for organization of the carbohydrate-dependent signaling domain involved in cellular differentiation of HL60 cells.

Human monoclonal anti-HCMV neutralizing antibody from phage display libraries

Human cytomegalovirus (HCMV) infection in immunocompromised patients causes considerable morbidity and mortality. Although ganciclovir prophylaxis reduces the incidence of HCMV disease, severe side effects raise serious problems. Thus, the development of new strategies for prophylaxis are clearly needed, and human monoclonal antibodies offer a potential alternative. We describe the cloning, using the phage display system, of a recombinant human Fab fragment against HCMV. A phage display library with 4 x 10(6) clones was panned three times against lysates of HCMV-infected cells, and screened by ELISA. Of six antigen-binding clones, one monoclonal antibody reacted strongly to HCMV. In immunostaining analysis, this Fab was able to stain HCMV-infected cells from 24 h post-infection (pi) through to 96 h pi, but not at 6 h pi. In the presence of cytosine arabinoside, HCMV-infected cells were not stained, even at 24 h pi. These results indicate that an HCMV protein that was recognized by the Fab was synthesized in the late phase of infection. In addition, this Fab exhibited neutralizing activity: at 1 microg/ml it reduced HCMV plaque formation by 50%. The Fab was able to neutralize three HCMV strains, but it did not neutralize HSV-1 or -2 infection.

Expression of early virus functions in human cytomegalovirus infected HEL cells: effect of ultraviolet light-irradiation of the virus.

Ultraviolet (u.v.) light-irradiation of human cytomegalovirus (HCMV) resulted in differential inactivation of virus capacities, e.g. induction of cell rounding, early antigens (EA), nuclear inclusion, HCMV DNA synthesis, cellular DNA synthesis, HCMV-specific DNA polymerase, cellular DNA polymerases and plaque production, while the capacity of HCMV to penetrate cell nuclei was not critically impaired. These results indicated that the virus-coded functions expressed after infection were responsible for sll these events except for HCMV-induced stimulation of cellular RNA synthesis which was enhanced by irradiation of the virus at a low dose of u.v. light (6600 ergs/mm2). In these experiments phosphonoacetic acid was effectively utilized to detect EA formation by immunofluorescent staining and to differentiate cellular DNA synthesis from virus DNA synthesis.

Bacterial expression of a human recombinant monoclonal antibody Fab fragment against hepatitis B surface antigen

The Fab fragment was cloned from the monoclonal cell line TAPC301‐CL4, which was produced using the Epstein‐Barr virus (EBV) transformation method. This cell line produces a human monoclonal antibody (CL4MAb) against the hepatitis B surface antigen (HBsAg). This MAb was shown to have hepatitis B virus (HBV) neutralizing activity in chimpanzees. The Fab fragment was produced by subjecting the heavy and light chain antibody genes of the TAPC301‐CL4 cell line to reverse transcription‐polymerase chain reaction, cloning the products in the plasmid vector pFab1‐His2 and introducing the plasmid into bacteria. Sequence analyses of the CL4Fab fragment revealed that the light and heavy chains belong to the Vk3a and VH3 groups of the immunoglobulin (Ig) family, respectively. An enzyme‐linked immunosorbent assay confirmed that specificity of the recombinant CL4Fab antibody against HBsAg was the same as that of the parental MAb. Flow cytometric analysis using PLC/PRF/5 (Alexander) cells, which express HBsAg, showed the reactivities of the CL4MAb and CL4Fab antibody were the same. These results suggest that the recombinant CL4Fab antibody produced by Escherichia coli using the new vector‐primer system developed for human IgG Fab fragments has a very high affinity for the HBsAg and may be useful clinically. A source for generation of human MAb for human therapy with very stable and specific expression was thus produced by isolating antibodies from EBV‐transformed cell lines. J. Med. Virol. 58:338–345, 1999.

Induction of Pre-Early Nuclear Antigen(s) in HEL Cells Infected with Human Cytomegalovirus

Human cytomegalovirus (HCMV)‐specific nuclear antigen could be detected within 1 hr after infection in human embryo lung cells by the anticomplement immunofluorescence (ACIF) test. This antigen has been named the pre‐early nuclear antigen (PENA) in this paper. Serum absorption tests suggested that PENA is immunologically different from the early antigen and the major nuclear inclusion antigens detected by the indirect immunofluorescence test before and after viral DNA replication, respectively. PENA‐forming ability of the virus corresponded to its plaque forming ability. PENA formation was not affected by phosphonoacetate but was inhibited by the addition of inhibitors of RNA and protein syntheses or by UV‐irradiation of infecting virus, suggesting that the formation of PENA depends on the expression of infecting virus gene functions. Virus‐specific proteins were isolated by indirect immunoprecipitation from HCMV‐infected cells exposed to 35S‐methionine. SDS‐polyacrylamide gel electrophoresis of the immunoprecipitate showed that at least two species of virus‐specific polypeptides with molecular weights of 70,000 and 30,000 were synthesized de novo within 3 hr after infection.

Recombinant antibody Fab against the hypervariable region 1 of hepatitis C virus blocks the virus adsorption to susceptible cells in vitro

Antibodies against hypervariable region 1 (HVR1) of hepatitis C virus (HCV) are putatively considered to be neutralizing. We previously found that monoclonal antibodies (mAbs) (30F1 and 30F3) against the HVR1 of HCV neutralize HCV in vitro. To develop potentially therapeutic molecules against HCV, we cloned cDNAs of antibody Fab fragments from the mouse hybridoma cells secreting these two mAbs. Fab fragments produced in Escherichia coli were purified by a single step of nickel-chelate affinity chromatography via a hexa-histidine tag. The specificity of the Fabs was confirmed by competition ELISA, BIAcore analysis, and N-terminal amino acid sequencing. The binding constant for the interaction with HVR1 was 1.39 nM for Fab 30F1 and 3.96 nM for Fab 30F3. The HCV capture assay and inhibition of HCV adsorption test demonstrated that both Fabs had neutralizing activity. The data may be useful for designing immunological therapy of HCV.

Morphogenesis of Nuclear Inclusions and Virus Capsids in HEL Cells Infected with Temperature-sensitive Mutants of Human Cytomegalovirus

The morphogenesis of nuclear inclusions and virus capsids in human embryonic lung cells infected with ts mutants of human cytomegalovirus at permissive (34 degrees C) and non-permissive (39 degrees C) temperatures was studied by indirect immunofluorescence (IF) and electron microscopic analyses and compared with the morphogenesis of these structures in wild-type virus infection with or without phosphonoacetate. Mutants tested belonged to five different complementation groups: two groups were DNA- (those unable to synthesize virus DNA at 39 degrees C) and the others were dna+. Based on the previous finding that the electron-dense, reticular nuclear inclusions (EM-NI) observed by the thin-section analysis correspond with nuclear inclusions (IF-NI) detected by the indirect IF staining (i.e. they occupy the same space in the nucleus), the following conclusions were obtained in ts mutant infection at 39 degrees C: (i) the formation of EM-NI, IF-NI and virus capsids requires replication of virus DNA. (II) The formation of EM-NI is not necessarily accompanied by the formation of IF-NI; EM-NI itself is not IF-positive unless it acquires virus-specific late antigens. (iii) The assembly of virus capsids occurs only in those cells in which EM-NI is formed; however, it can occur without the formation of IF-NI. (iv) Virus capsids assembled are not the major antigens responsible for the fluorescence of nuclear inclusions.

Inhibition of HIV replication by a CD4‐reactive Fab of an IgM clone isolated from a healthy HIV‐seronegative individual

HIV replication is restricted by some anti‐CD4 mouse mAb in vitro and in vivo. However, a human monoclonal anti‐CD4 Ab has not been isolated. We screened EBV‐transformed peripheral B cells from 12 adult donors for CD4‐reactive Ab production followed by functional reconstitution of Fab genes. Three independent IgM Fab clones reactive specifically to CD4 were isolated from a healthy HIV‐seronegative adult (∼0.0013% of the peripheral B cells). The germ line combinations for the VH and VL genes were VH3‐33/L6, VH3‐33/L12, and VH4‐4/L12, respectively, accompanied by somatic hypermutations. Genetic analysis revealed a preference for V‐gene usage to develop CD4‐reactive Ab. Notably, one of the CD4‐reactive clones, HO538‐213, with an 1×10−8 M dissociation constant (Kd) to recombinant human CD4, limited the replication of R5‐tropic and X4‐tropic HIV‐1 strains at 1–2.5 μg/mL in primary mononuclear cells. This is the first clonal genetic analysis of human monoclonal CD4‐reactive Ab. A mAb against CD4 isolated from a healthy individual could be useful in the intervention of HIV/AIDS.