Biomedical subjects

I Takashima

Publications and source records attributed to I Takashima.

At least 19 recordsLinked to original sources

PubMed2001

Voltage-sensitive dye versus intrinsic signal optical imaging: comparison of optically determined functional maps from rat barrel cortex.

Using intrinsic and voltage-sensitive dye optical imaging methods, somatosensory-evoked neural activity and the consequent metabolic activity were visualized in the barrel cortex at high temporal and spatial resolution. We compared maps of neural and metabolic activity from the perspective of spatial distribution in the cortex. There was good agreement between the two functional maps, if the extent of metabolic activity before a prominent increase in cerebral blood volume (CBV) was assessed. This result indicates that oxygen consumption occurs before CBV changes, in approximately the same cortical area as that in which the preceding neural activity was evoked. This also suggests that the intrinsic signal reflects subthreshold synaptic activity, as well as spiking activity, which is similar to the dye-related signals.

Afferent Pathways↗

PubMed2001

Evaluation of European tick-borne encephalitis virus vaccine against recent Siberian and far-eastern subtype strains.

To evaluate the efficacy of the European TBE vaccine in east-Siberian and far-eastern regions of Russia, we examined the immune responses of the vaccine against recent TBE virus Siberian (Irkutsk) and far-eastern (Khabarovsk and Vladivostok) isolates. The sera of vaccinated humans showed efficient neutralizing antibody titers (> or =20) against Siberian and far-eastern strains. To evaluate the efficacy of the vaccine in vivo, mice were vaccinated and challenged with lethal doses of the viruses. All vaccinated mice survived each virus challenge. These results suggest that the European vaccine can prevent the TBE virus infection in east-Siberian and far-eastern regions of Russia.

Adult↗

PubMed2001

Pallidal activity is involved in visuomotor association learning in monkeys.

In order to examine whether the basal ganglia are involved in arbitrary visuomotor association, we recorded neuronal activity in the internal segment of the globus pallidus (GPi) of monkeys during a conditional visuomotor learning task. Two monkeys were presented a cueing visual stimulus, and following a delay period required to push, pull or turn a manipulator according to the cue. GPi neurons showed changes in activity during the delay period when the animals performed the task on the basis of a familiar stimulus-response association. Those changes in delay activity were enhanced as the monkeys were learning a new visuomotor association. The enhancement of the changes was selective to a following response. These results suggest that the basal ganglia are involved in arbitrary visuomotor association, especially during the learning of new associations.

Animals↗

PubMed2001

Truncated hantavirus nucleocapsid proteins for serotyping Hantaan, Seoul, and Dobrava hantavirus infections.

Truncated recombinant nucleocapsid proteins (rNPs) of Hantaan virus (HTNV), Seoul virus (SEOV), and Dobrava virus (DOBV) were expressed by a baculovirus system. The truncated rNPs, which lacked 49 (rNP50) or 154 (rNP155) N-terminal amino acids of the NPs of HTNV, SEOV, and DOBV, were able to differentiate HTNV-, SEOV-, and DOBV-specific immune sera. Recombinant NP50s retained higher reactivities than rNP155s and were proven useful for enzyme-linked immunosorbent assay (ELISA). The ELISAs based on the rNP50s of HTNV, SEOV, and DOBV successfully differentiated three groups of patient sera, previously defined by neutralization tests: 17 with HTNV infection, 12 with SEOV infection, and 20 with DOBV infection. The entire rNP of Puumala virus (PUUV) distinguished PUUV infection from the other types of hantavirus infection. Serotyping with these rNP50s can be recommended as a rapid and efficient system for hantavirus diagnosis.

Animals↗

PubMed2001

Characterization of in vitro and in vivo antiviral activity of lactoferrin and ribavirin upon hantavirus.

Mechanisms of anti-hantaviral activities of bovine lactoferrin (LF) and ribavirin (Rbv) were investigated. Hantavirus focus formation at 48 hr was 15% of the control in cells treated with 400 microg/ml LF for 1 hr at 37 degrees C prior to viral infection. Post infection treatment with 100 microg/ml Rbv also inhibited the focus formation to 2.5% of the control. Combined LF pre- and Rbv post-infection treatment completely inhibited focus formation. Viral glycoprotein (G2) and nucleocapsid protein (NP) syntheses were delayed in LF pretreated cells up to 24 hr post infection (hpi) but became comparable to the control by 48 hpi. Further, LF inhibited viral shedding at 24 hpi but did not inhibit shedding after 48 hpi. However, Rbv was able to inhibit synthesis of viral proteins, (+) and (-) strand RNAs also inhibited viral shedding after 24 hr. These results suggest that LF inhibits viral adsorption to cells, while Rbv inhibits viral RNA synthesis. For in vivo trials of LF and Rbv, LF pre- and Rbv post-treatment were evaluated in suckling mice infected with hantavirus, of which 7% survived. LF concentrations of 40 and 160 mg/kg administered prior to viral challenge improved survival rates to 15% and 70%, respectively for single administration and 85% and 94%, respectively, for double administration. Rbv concentrations of 25 and 50 mg/kg gave survival rates of 68% and 81%, respectively. This suggests that both LF and Rbv are efficacious in hantavirus infection in vivo.

Animals↗

PubMed2001

Epidemiology of tick-borne encephalitis (TBE) and phylogenetic analysis of TBE viruses in Japan and Far Eastern Russia.

In Oshima, the southern part of Hokkaido, a tick-borne encephalitis (TBE) patient was found in 1993; in addition TBE virus was isolated from the blood samples of sentinel dogs, ticks pools, and rodents spleens in 1995 and 1996 by suckling mice. To identify when these TBE viruses emerged in Hokkaido, the times of divergence of TBE virus strains isolated in Oshima and Far Eastern Russia were estimated. TBE virus was isolated in Khabarovsk in 1998, and the nucleotide sequences of viral envelope protein genes of isolates from Oshima and Khabarovsk were compared. Based on the synonymous substitution rates of these virus E-protein genes, the lineage-divergence times of these TBE virus strains were predicted phylogenetically to be approximately 260-430 years ago. Furthermore, the virulence of TBE virus isolates from Oshima and Khabarovsk were compared in a mouse model. The results showed that the isolates possessed very similar virulence in mice. European TBE vaccine was found to be effective in TBE virus, Hokkaido strain. This review provides evidence that the Oshima strains of TBE virus in Hokkaido emerged from the Far Eastern Russia a few hundred years ago, which explains why the virulence of these strains is similar to that of TBE viruses isolated in Russia. Practical application of the vaccine should be considered in Japan.

Amino Acid Sequence↗

PubMed2001

Epizootiological survey of hantavirus among rodent species in Ningxia Hui Autonomous Province, China.

Hantaviral antibodies were detected in the sera from Apodemus (A.) agrarius and A. peninsulae captured in Ningxia province, China by several different serological diagnostic methods. A total of 409 sera from rodent and insectivore species were collected in 1999 and examined by indirect immunofluorescent antibody assay (IFA). Among them, 19 of 191 (9.9%) sera of A. agrarius and 1 of 13 (7.7%) sera of A. peninsulae were positive for hantaviral antibodies. The other species (Rattus norvegicus, Mus musculus, Cricetulus triton, and Sorex cylindricauda) were negative. The reaction pattern of positive serum was characterized as scattered and granular virus antigens in the cytoplasm of hantavirus infected Vero E6 cells. Some of the A. agrarius sera positive for hantavirus were further examined by Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and the focus reduction neutralization test (FRNT). By WB, positive sera showed the same specific reaction pattern of baculovirus-expressed recombinant hantaviral nucleocapsid protein, as shown in hantavirus-immune serum. By ELISA, IFA-positive sera showed significantly higher optical densities (around 1.0) than the negative A. agrarius sera. Hantaan type hantavirus was neutralized with the positive sera. These results suggest that A. agrarius have hantavirus infection and may play a role as a reservoir animal for hantavirus in Ningxia Hui Autonomous Province, China.

Animals↗

PubMed2000

In vitro antiviral activity of lactoferrin and ribavirin upon hantavirus.

Bovine lactoferrin (LF) and ribavirin (Rbv) were tested as antiviral agents against Seoul type hantavirus (SR-11 strain) in vitro. Hantaviral foci number in Vero E6 cells infected with SR-11 was reduced with LF treatment by 5 days post infection to obtain a 50% effective dose (ED50) of 2500 microg/ml, while pretreatment with LF was highly efficacious having an ED50 of 39 microg/ml. Conversely, 1 h pretreatment with Rbv revealed no inhibition of viral focus formation but could significantly reduce the number of viral foci (ED50: 10 microg/ml) when used from the time of viral infection. One hour pre-treatment of the cell monolayer with LF and subsequent addition of Rbv revealed a synergistic anti-hantaviral effect against SR-11, <20 FFU/ml as compared to 10(5) foci/ml in the control. One hour treatment of SR-11 with LF prior to cell inoculation gave an ED50 of 312.5 microg/ml. Whereas, washing the LF-pretreated cell monolayer with PBS demonstrated minimal focus reduction, suggesting LF lightly adheres to cells. These results indicate that LF has anti-hantaviral activity in vitro and inhibition of virus adsorption to cells which play an important role in revealing the anti-hantaviral activity of LF. This paper reports for the first time the anti-hantaviral effect of LF.

Animals↗

PubMed2000

Detection of hantaviral antibodies among patients with hepatitis of unknown etiology in Japan.

Hantaviral antibodies were detected in the sera from patients with hepatic disease of unknown etiology in Japan by several different serological diagnostic methods. A total of 105 sera from diseased patients which were negative to A-G hepatitis virus infections in the Tokyo area were tested. Among them, 3 out of 73 sera from patients with chronic hepatic disease were positive to hantaviral antibody by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent antibody assay (IFA) and Western blot analysis (WB). Neutralizing antibody titers of the 3 sera to Seoul virus (SEO) were 4 to 8 times higher than those to Hantaan virus (HTN). However, all of the 32 sera from patients with acute hepatitis were negative for hantaviral antibody. Among the 60 patients with chronic hepatitis in Hokkaido which were serologically negative to B and C hepatitis virus infection, one was positive for hantaviral antibody by ELISA and WB. In contrast, the sera from healthy adults in Japan, 550 from the Honshu and Kyushu regions, and 1,000 from the Hokkaido region, were negative for hantavirus antibody. These results show that hantaviral antibodies are more frequently detected in patients with hepatic disease than in healthy adults. However, the observation that no positive sera were detected from patients with acute hepatitis implies that hantavirus might not be directly related to hepatitis.

Acute Disease↗

PubMed2000

Characterization of monoclonal antibodies against Hokkaido strain tick-borne encephalitis virus.

A tick-borne encephalitis (TBE) patient was found in Hokkaido in 1993, and TBE viruses were isolated from animals and ticks in our previous studies. To develop a diagnostic reagent to identify TBE viruses, monoclonal antibodies (Mabs) were produced against the TBE virus strain Hokkaido (Oshima 5-10). Seven Mabs were obtained which reacted with the envelope protein of the Oshima 5-10 strain. These Mabs were flavivirus genus-specific, TBE virus complex-specific or TBE virus type-specific. The Mabs are applicable for identification of TBE virus strains.

Animals↗

PubMed2000

The mechanism of actinomycin D-mediated increase of Borna disease virus (BDV) RNA in cells persistently infected by BDV.

The transcriptional mechanism of Borna disease virus (BDV) has been poorly understood. We have analyzed transcription of the virus upon various stimuli in Madin-Darby canine kidney cells which were persistently infected by BDV (MDCK/BDV). Treatment with actinomycin D (ActD) increased the level of BDV RNA, shifting the size of RNA from 1.9 kb to 2.3 kb beginning 5 hr after the treatment. To understand the mechanism of this unique modulation of BDV RNA, we conducted several experiments. The RNA increase occurred at the stage in which synthesis of cellular intrinsic mRNA was intact, suggesting BDV does not compete with cellular transcriptional machinery for intrinsic RNA polymerase II. The BDV transcription was also enhanced by cycloheximide treatment, indicating that newly synthesized viral or cellular proteins are not necessary for viral transcription. However, a shift in the RNA size was not observed for cycloheximide-induced BDV RNA. The increase in viral transcription persisted during the cellular apoptotic process consequent to p53 gene accumulation beginning 1 hr after ActD treatment. Caspase inhibitors Z-VAD and DEVD-CHO repressed the apoptotic process but failed to block the increase in BDV transcription. In addition, adenovirus-mediated transduction of wild-type p53 did not alter the BDV transcription, indicating that the increase in BDV transcription was independent of the p53-mediated apoptotic process. Other various stimuli that evoke cellular signal transductions failed to alter BDV transcription. Agents inhibitory to topoisomerase except adriamycin failed to enhance BDV transcription, indicating that the increase in BDV transcription is not mediated by an inhibitory action to the topoisomerase II of ActD. Adriamycin showed an increase and size-shift of BDV RNA similar to ActD. These results suggest that intercalation of the viral genome itself with ActD is related to the stabilization of viral RNA and alteration of RNA size rather than secondary host cell changes.

Animals↗

PubMed2000

Pathogenicity of Hantaan virus in newborn mice: genetic reassortant study demonstrating that a single amino acid change in glycoprotein G1 is related to virulence.

Two Hantaan virus strains, clone 1 (cl-1), which is virulent in newborn mice, and its attenuated mutant (mu11E10), were used to examine the pathogenesis of Hantaan virus infection in a mouse model and identify virus factors relating to virulence. After subcutaneous inoculation of newborn BALB/c mice, cl-1 caused fatal disease with high viral multiplication in peripheral organs, but mu11E10 produced nonfatal infection with a low level of virus multiplication. Intracerebral inoculation of either strain caused fatal disease. Histopathological changes in the dead animals were prominent in the brain, indicating that the brain is the target organ and produces the fatal outcome. These results indicate that mu11E10 has a generally less virulent phenotype, and because of decreased multiplication in peripheral tissues, neuroinvasiveness is also decreased. An experiment with genetic reassortant viruses showed that in newborn mice the M segment is the most related to virulence and the L segment is partly related. Sequence comparison detected a single deduced amino acid change (cl-1 Ile to mu11E10 Thr) at amino acid number 515 in glycoprotein G1. One nucleotide change, but no amino acid substitution, was observed in the noncoding region of the L segment. In mouse brain microvascular endothelial cells in vitro, viruses possessing a cl-1-derived M segment grew more rapidly than viruses containing a mu11E10-derived M segment. These results suggest that the single amino acid change in the glycoprotein alters peripheral growth, which affects invasion of the central nervous system in mice.

Amino Acid Substitution↗

PubMed2000

Optical recording study of granule cell activities in the hippocampal dentate gyrus of kainate-treated rats.

In the epileptic hippocampus, newly sprouted mossy fibers are considered to form recurrent excitatory connections to granule cells in the dentate gyrus and thereby increase seizure susceptibility. To study the effects of mossy fiber sprouting on neural activity in individual lamellae of the dentate gyrus, we used high-speed optical recording to record signals from voltage-sensitive dye in hippocampal slices prepared from kainate-treated epileptic rats (KA rats). In 14 of 24 slices from KA rats, hilar stimulation evoked a large depolarization in almost the entire molecular layer in which granule cell apical dendrites are located. The signals were identified as postsynaptic responses because of their dependence on extracellular Ca(2+). The depolarization amplitude was largest in the inner molecular layer (the target area of sprouted mossy fibers) and declined with increasing distance from the granule cell layer. In the inner molecular layer, a good correlation was obtained between depolarization size and the density of mossy fiber terminals detected by Timm staining methods. Blockade of GABAergic inhibition by bicuculline enlarged the depolarization in granule cell dendrites. Our data indicate that mossy fiber sprouting results in a large and prolonged synaptic depolarization in an extensive dendritic area and that the enhanced GABAergic inhibition partly masks the synaptic depolarization. However, despite the large dendritic excitation induced by the sprouted mossy fibers, seizure-like activity of granule cells was never observed, even when GABAergic inhibition was blocked. Therefore, mossy fiber sprouting may not play a critical role in epileptogenesis.

Animals↗

PubMed2000

Co-transduction of herpes simplex virus thymidine kinase gene and human interleukin-2 gene into mouse ovarian cancer cell line, OVHM.

We studied the thymidine kinase (TK) gene and the interleukin (IL)-2 gene co-transduction into tumor cells for a possible strategy of cancer gene therapy. A murine ovarian cancer cell line, OVHM, was retrovirally transduced with the TK (OVHM/TK) or the IL-2 gene (OVHM/IL-2). The TK or IL-2 expression was permanent in OVHM/TK or OVHM/IL-2. OVHM/TK cells were susceptible to gancyclovir (GCV) in vitro, and their intraperitoneal growth was completely regulated with GCV administration. The bystander effect was not observed in vitro and in vivo in this model, and only the marginal emergence of immune involvement was observed in the OVHM/TK-cured mice with GCV. OVHM/IL-2 cells produced IL-2 biologically active to be immunogenic, but still tumorigenic to kill the mice when inoculated intraperitoneally. Then, OVHM/TK cells were co-transduced with the IL-2 gene to establish OVHM/TK/IL-2 cells. OVHM/TK/IL-2 cells were also susceptible to GCV and transiently produced active IL-2. A significant resistance against the challenge of parental tumor cells was observed in the mice that were inoculated with OVHM/TK/IL-2 cells and cured with GCV administration. It is suggested that tumor cells transduced with both TK and IL-2 genes could be regressed with GCV administration with subsequent generation of immune activation in the host. Since the bystander effect may not always be a common phenomenon in gene therapy using the TK gene, this type of combination may be advantageous in the clinical application of gene therapy for human cancers.

Animals↗

PubMed1999

High-speed CCD imaging system for monitoring neural activity in vivo and in vitro, using a voltage-sensitive dye.

We have designed and constructed a high-speed CCD imaging system for optically detecting neural activity from preparations stained externally with a voltage-sensitive dye, and have used this system to image evoked and epileptiform neural activity in the rat somatosensory cortex. The imaging system uses a commercially available 1/3-in. CCD chip, and it can continuously capture images for more than 8 s, at 1000 frames/s, with a spatial resolution of 128 x 62 pixels. The spatial/temporal resolution of the CCD sensor is variable by changing the geometry of on-chip binning pixels, which can be controlled by a PC/AT computer. Dye bleaching correction was not necessary for long-term imaging of epileptiform neural events, since the sensitivity of the CCD sensor was increased by combining the signal from adjacent pixels.

Animals↗

PubMed1999

Optical recording of cortical activity after in vitro perfusion of cerebral arteries with a voltage-sensitive dye.

Cortical neuronal architecture and connectivity can be analyzed with high-resolution optical imaging after staining the in vitro isolated guinea pig brain preparation by circulating the voltage-sensitive dye RH795 via the arterial system. To establish this new technique, electrical field potentials evoked in the piriform and entorhinal cortices by lateral olfactory tract stimulation were correlated to the optical signal. The depth analysis of the optical response was performed by evaluating the contribution of the mono- and poly-synaptic components of the signal generated in different layers after applying a pair-pulse stimulation protocol. The tangential propagation of neuronal activity in olfactory cortices was evaluated by gathering several 4.2 x 4.2 mm images recorded from adjacent cortical areas. The real-time optical imaging technique applied to the isolated guinea pig brain can be successfully utilized to study the integrative properties of cortical neurons ensembles.

Animals↗

PubMed1999

Protection against tick-borne encephalitis virus isolated in Japan by active and passive immunization.

In order to establish a firm preventive measure for tick-borne encephalitis (TBE) in Japan, we evaluated the immune response of European vaccine against Japanese TBE virus strain (Oshima 5-10) for man and mouse. Furthermore, the efficacy of pre- and post-exposure protection by a polyclonal rabbit anti-TBE virus serum was examined in the mouse model. 80% of vaccinees seroconverted against Oshima 5-10 strain after the 2nd immunization of vaccine and the remaining 20% seroconverted after the 3rd immunization. Two persons with pre-existing anti-Japanese encephalitis virus (JEV) antibodies showed low immune responses against TBE virus. In mouse vaccination and challenge tests, efficient protection was observed in mice challenged with lethal doses of Oshima 5-10 strain as well as those observed in mice with the Western subtype and the Far Eastern subtype of TBE strains. Pre-exposure treatment with rabbit anti-TBE virus serum provided complete protection against lethal challenge with Oshima 5-10 strain. For post-exposure treatment with the antibody, significant protection was observed when mice were treated 24 h after virus challenge, whereas it was not observed 48 h after virus challenge. reserved.

Animals↗

PubMed1999

Pathogenicity of tick-borne encephalitis virus isolated in Hokkaido, Japan in mouse model.

The pathogenic characteristics of tick-borne encephalitis (TBE) virus strain (Oshima 5-10) isolated from a sentinel dog in Hokkaido, Japan, was compared by use of a mouse model with several inoculation routes to other strains of TBE virus (the Far Eastern subtype; Sofjin strain and the Western subtype; Hochosterwitz strain) and TBE complex virus (Langat virus; TP-21 strain). The degree of neuroinvasiveness of the strains in mice subcutaneously (s.c.) inoculated was Sofjin equaled Hochosterwitz which was greater than Oshima and TP-21, respectively. Neurovirulence, as determined after intracerebral inoculation was Sofjin > Oshima = Hochosterwitz > TP-21. Virus replication in the brains of mice s.c. or intracerebrally inoculated with Oshima strain was slower and of lower titer than that of Sofjin strain. Histopathological findings indicated that subarachnoid infiltration of mononuclear cells prior to necrosis of the cerebrum was characteristic in Oshima strain. These findings indicated that the Oshima strain possessed a pathogenic potential common to TBE viruses and is less virulent for mice as compared with the two other TBE strains examined.

Animals↗