Biomedical subjects

Isao Nagano

Publications and source records attributed to Isao Nagano.

At least 37 records · Page 2Linked to original sources

PubMed2004

Molecular expression of a cysteine proteinase of Clonorchis sinensis and its application to an enzyme-linked immunosorbent assay for immunodiagnosis of clonorchiasis.

We produced a recombinant cysteine proteinase of Clonorchis sinensis and tested its value as an antigen for serologic diagnosis of C. sinensis infections. The predicted amino acid sequence of the cysteine proteinase of C. sinensis was 58, 48, and 40% identical to those of cathepsin L cysteine proteinases from Paragonimus westermani, Schistosoma japonicum, and Fasciola hepatica, respectively. Western blotting analysis showed that sera from patients infected with C. sinensis strongly reacted with the recombinant protein and that sera from patients infected with S. japonicum weakly reacted with the recombinant protein. Antibody against the recombinant protein stained proteins migrating at about 37 and 28 kDa in C. sinensis adult worm crude extracts. Immunostaining revealed that the cysteine proteinase of C. sinensis was located in the intestinal epithelial cells of the adult parasite and in intrauterine eggs. The specificity and sensitivity of the recombinant antigen or C. sinensis adult worm crude extracts were assessed by an enzyme-linked immunosorbent assay (ELISA) using serum samples from humans infected with different parasites, including 50 patients with clonorchiasis, and negative controls. The sensitivities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96 and 88%, respectively. The specificities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96.2 and 100%, respectively. The results suggested that the recombinant cysteine proteinase-based ELISA could provide a highly sensitive and specific assay for diagnosis of clonorchiasis.

Amino Acid Sequence↗

PubMed2004

Extension of ischemic therapeutic time window by a free radical scavenger, Edaravone, reperfused with tPA in rat brain.

3-methyl-1-phenyl-2-pyrazolin-5-one (Edaravone) is a free radical scavenger. We tested the hypothesis that combination treatment of Edaravone and recombinant tissue plasminogen activator (tPA) extends the therapeutic time window. Male Wistar rats were subjected to 1.5-, 3.0- or 4.5-hour middle cerebral artery (MCA) occlusion (MCAO) by a nylon thread. Animals were randomly divided into four groups. The Sham group rats were operated without MCAO and drug injection. In the Vehicle-treated group the same volume of saline was given every 1.5 hours from just after MCAO to just before reperfusion. In the Vehicle + tPA-treated group saline injection was given as above and tPA (5 mg/kg, i.v.) was given once just after reperfusion. Edaravone+tPA-treated group: Edaravone (3 mg/kg, i.v.) was given every 1.5 hours instead of saline and tPA injection as above. Survival rate, infarct size and evidence of apoptosis and hemorrhage were examined in the animals. Combining administration of Edaravone+tPA significantly increased survival rate after 3 hours of transient MCAO, and reduced infarct volume after 1.5 hours of transient MCAO compared with the vehicle or vehicle+tPA groups. In Edaravone+tPA-treated group, the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and 4-hydroxynonenal (4-HNE) positive cells were reduced at 16 hours after 3 hours of transient MCAO, but not in advanced glycation end products (AGEs) and 8-hydroxy-2'-deoxyguanosine (8-OHdG). Hemorrhage rate and the area decreased in the Edaravone+tPA-treated group. The combination therapy of Edaravone+tPA increased survival rate, and reduced the infarct volume and hemorrhage with reduction of lipid peroxidation. Therefore, Edaravone combination is expected to extend the therapeutic time window of tPA in the clinical situation.

Aldehydes↗

PubMed2004

[Neurosyphilis].

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Anti-Bacterial Agents↗

PubMed2004

[A case of cortical infarction showing unilateral facial and hypoglossal paresis].

We describe a case presenting with facial and hypoglossal nerve paresis due to cortical cerebral infarction. A 54-year-old man visited our hospital complaining of sudden episode of dysarthria and facial paresis. Neurological findings revealed tongue deviation to the left and left facial paresis with forehead wrinkling while neither limb paralysis nor sensory impairment was observed. Head CT performed on day 3 after onset revealed a cortical infarction in the right prefrontal gyrus. Symptoms gradually improved with medical management. This patient had isolated facial and hypoglossal nerve paresis without other neurological symptoms. Patients with mild paresis of cranial nerves should be diagnosed carefully, because their paresis could be supranuclear type.

Cerebral Infarction↗

PubMed2004

[A case of subacute myelitis associated with primary Sjögren syndrome showing no MRI abnormality and diagnosed by somatosensory evoked potentials].

We describe a case of Sjögren syndrome presenting with subacute transverse myelopathy that improved in response to steroid therapy. A 55-year-old woman with a history of NIDDM developed progressive sensory disturbance and weakness of the both legs, and micturition disturbance during a 6-month period, resulting in walking difficulty. On admission, neurological examination revealed paraplegia with extensor plantar reflex and sensory disturbance below Th 6 level. Lumbar puncture disclosed a normal cell count and a mild elevation of protein in the CSF. As subacute myelitis at the level of Th 6 was suspected, spinal plain and enhanced MRI was performed, which revealed no abnormalities. In contrast, tibial somatosensory evoked potential (SEP) could not be evoked while median SEP was almost normal, indicating thoracic cord involvement. She was diagnosed with primary Sjögren syndrome based on xerostomia, apple-tree appearance in parotid sialography, mononuclear cell infiltration in the salivary gland biopsied, and elevated anti-SS-A and SS-B antibody titers. We considered that her myelopathy was associated with Sjögren syndrome, and started steroid pulse therapy. After three courses of steroid pulse therapy followed by oral prednisolone, she showed clinical and SEP improvements. It is conceivable that the present case may represent Sjögren syndrome complicated with myelopathy that shows no MRI abnormality and well responds to steroid therapy. SEP can be a valuable tool for assessing the presence and severity of spinal cord involvement, and the efficacy of treatments.

Evoked Potentials, Somatosensory↗

PubMed2003

Sustained induction of survival p-AKT and p-ERK signals after transient hypoxia in mice spinal cord with G93A mutant human SOD1 protein.

Expression of survival p-AKT and p-ERK signals was examined by immunohistochemistry and Western blotting in the lumbar spinal cord of 12-week-old presymptomatic mice with human mutant G93A SOD1 gene (transgenic, Tg) and their wild-type (Wt) littermates during normoxia, and 0 and 6 h after 2 h of 9% hypoxia. During normoxia, a stronger p-AKT signal was detected in the nucleus of the motor neurons of Tg animals. At 0 h of recovery from 2 h of hypoxia, both p-AKT and p-ERK signals were induced at a slightly lower level in Tg (1.1-1.2-fold) compared to those of Wt (1.2-1.5-fold) animals. At 6 h of recovery, both p-AKT and p-ERK signals were sustained in the lumbar spinal motor neurons of Tg animals, while those in Wt animals quickly returned to baseline level. As a control, at 6 h of recovery, the hippocampus of Tg animals showed significantly sustained p-AKT levels, but not p-ERK levels, compared to Wt. The current results suggest that the presence of mutant SOD1 alters survival p-AKT and p-ERK signals, possibly to compensate for the acquired gain-of-function of the mutant protein.

Amino Acid Substitution↗

PubMed2003

Hypoxic induction of vascular endothelial growth factor is selectively impaired in mice carrying the mutant SOD1 gene.

Localization and hypoxic induction of vascular endothelial growth factor (VEGF) was examined in the spinal cord of transgenic mice carrying a mutation in the superoxide dismutase 1 gene. Immunohistochemical and immunofluorescent study demonstrated that VEGF is mainly expressed in motor neurons before and after hypoxia. Baseline expression of VEGF was higher in transgenic (Tg) mice than in wild-type (Wt) littermates. However, VEGF was hardly induced after hypoxia in Tg mice, whereas Wt mice showed an approximate nine-fold increase. Impaired VEGF induction was evident in Tg mice at 12 weeks of age, when they were still presymptomatic. In contrast, baseline and hypoxic expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor did not differ between Tg and Wt mice. Thus, the present study demonstrates that hypoxic induction of VEGF in Tg mice is selectively impaired from a very early stage, suggesting profound involvement in the pathogenesis of motor neuron degeneration in this animal model of amyotrophic lateral sclerosis.

Animals↗

PubMed2003

Spatiotemporal changes of apolipoprotein E immunoreactivity and apolipoprotein E mRNA expression after transient middle cerebral artery occlusion in rat brain.

Apolipoprotein E (ApoE) is a constituent of lipoprotein and plays an important role in the maintenance of neural networks. However, spatiotemporal differences in ApoE expression and its long-term role in neural process after brain ischemia have not been studied. We investigated changes of ApoE immunoreactivity and ApoE mRNA expression both in the core and in the periischemic area at 1, 7, 21, or 56 days after 90 min of transient middle cerebral artery occlusion. Double stainings for ApoE plus NeuN or plus ED1 were performed in order to identify cell type of ApoE-positive stainings. The maximal increase of ApoE expression was observed at 7 days in the core and at 7 and 21 days in the periischemic area. In the core, ApoE plus NeuN double-positive cells increased at 1 and 7 days, without ApoE mRNA expression, whereas they increased in the periischemic area, with a peak at 21 days, with ApoE mRNA expression in glial cells but not in neurons. On the other hand, ApoE plus ED1 double-positive cells increased only in the core, with a peak in number at 7 and 21 days and marked ApoE mRNA expression in macrophages. The present study suggests that ApoE plays various important roles in different type of cells, reflecting spatiotemporal dissociation between degenerative and regenerative processes after brain ischemia, and that ApoE is profoundly involved in pathological conditions, such as brain ischemia.

Animals↗

PubMed2003

Cooperative expression of survival p-ERK and p-Akt signals in rat brain neurons after transient MCAO.

In order to determine possible coordinate expression of major survival signals, immunofluorescent analyses for phosphorylated ERK (p-ERK) and phosphorylated Akt (p-Akt) were carried out after 90 min of transient middle cerebral artery occlusion (MCAO) in rats. p-Akt single positive cells (E-/A+) were found in the sham control brains with weak signal intensity. The levels of both survival signals concurrently increased from 1 to 3 h after the reperfusion with the peak at 1 h, and the signals were much stronger in the ischemic penumbra (IP) than ischemic core (IC). The number of E-/A+ cells was larger in both the IC and IP than that of p-ERK single positive cells (E+/A-). The E+/A- cells were primarily expressed at 1 h in the IP. The number of p-ERK plus p-Akt double positive cells (E+/A+) peaked at 1 h, and the intensity was much stronger in the IP than IC. These findings suggest that p-ERK and p-Akt play independent roles, respectively as emergency or maintenance signal for survival at an early stage after reperfusion, and that both signals were cooperatively expressed especially in the IP.

Animals↗

PubMed2003

Dissociative increase of oligodendrocyte progenitor cells between young and aged rats after transient cerebral ischemia.

To investigate the effect of aging on the oligodendrocyte progenitor cells (OPCs) after cerebral ischemia, neuron-glia antigen 2 (NG2) chondroitin sulfate proteoglycan was examined at 1, 3 and 7 days after 90 min of transient middle cerebral artery occlusion in young and aged brains. The number of NG2 positive cells increased in the ischemic penumbra at 3 and 7 days after reperfusion, while those decreased in the ischemic core. At 7 days, the number of NG2 positive cells was significantly greater in the young than the aged brains, and the processes of NG2 positive cells enlarged and were highly branched in the young than the aged brains. These results suggest that the young brain showed a higher potential of proliferation and process branching of OPCs than the aged brains.

Aging↗

PubMed2003

Temporal and spatial changes of free cholesterol and neutral lipids in rat brain after transient middle cerebral artery occlusion.

In order to examine lipid metabolism in relation to neural process following brain ischemia, we investigated temporal and spatial changes of free cholesterol (FC) and neutral lipids (NLs) after 90 min of transient middle cerebral artery occlusion (MCAO). Filipin and Nile Red stainings were performed to detect mainly FC and NLs, respectively. Double stainings for Nile Red plus ED1, MAP2, or GFAP were performed in order to identify cell type of positive stainings. Filipin stanining decreased during 1-7 day and lost at 21 day after transient MCAO in the ischemic core, but did not change in the penumbra. Nile Red positive droplets reached the maximum at 7 day after transient MCAO and gradually decreased in the core, while the peak time delayed in the penumbra. MAP2 immunoreactivity lost at 7 day in the core, and increased in the penumbra during 7-56 day. Most Nile Red positive droplets were double positive for ED1 in the core, and were localized within GFAP positive cells in the penumbra. These results suggest that changes of FC and NLs are different temporally and spatially between the core and penumbra in relation to degenerative and regenerative neural processes following brain ischemia.

Animals↗

PubMed2003

Temporal profile of stem cell division, migration, and differentiation from subventricular zone to olfactory bulb after transient forebrain ischemia in gerbils.

The stage of neurogenesis can be divided into three steps: proliferation, migration, and differentiation. To elucidate their detailed relations after ischemia, the three steps were comprehensively evaluated, in the subventricular zone (SVZ) through the rostral migratory stream (RMS) to the olfactory bulb (OB), in adult gerbil brain after 5 minutes of transient forebrain ischemia. Bromodeoxyuridine (BrdU), highly polysialylated neural cell adhesion molecule (PSA-NCAM), neuronal nuclear antigen (NeuN), and glial fibrillary acidic protein (GFAP) were used as markers for proliferation, migration, and differentiation, respectively. The number of BrdU-labeled cells that coexpressed PSA-NCAM and the size of PSA-NCAM-positive cell colony increased in the SVZ with a peak at 10 d after transient ischemia. In the RMS, the number of BrdU-labeled cells that coexpressed PSA-NCAM increased, with a delayed peak at 30 d, when the size of RMS itself became larger and the number of surrounding GFAP-positive cells increased. In the OB, BrdU + NeuN double positive cells were detected at 30 and 60 d. NeuN staining and terminal deoxynucleotidyl dUTP nick-end labeling staining showed no neuronal cell loss around the SVZ, and in the RMS and the OB after transient ischemia. These findings indicate that transient forebrain ischemia enhances neural stem cell proliferation in the SVZ without evident neuronal cell loss, and has potential neuronal precursor migration with activation of GFAP-positive cells through the RMS to the OB.

Animals↗

PubMed2003

[A case of trichinellosis with spontaneous remission].

We experienced a case of trichinellosis that had traveled in Kenya. The initial symptoms of the patient were myalgia and fever which started 2 weeks after she came back from Kenya, and blood examination showed marked eosinophilia (14,300/mm3) and elevated creatinine kinase (826IU/L). When we made serological diagnosis of trichinellosis 3 weeks after onset, symptoms started to resolve spontaneously and we observed the patient without any medication. As of 2 months after onset, symptoms and abnormal findings in laboratory data almost disappeared. The patient was speculated to be infected with trichinella spp. by incompletely cooked wild animal meats including alligator, zebra, pig, and ostrich during travel in Kenya. Since high incidence of trichinella infection in both wild and domestic animals has been reported in some areas of developing countries, travelers must be aware that raw or incompletely cooked animal meats can be source of trichinella spp.

Animals↗

PubMed2003

Intraspecies polymorphism of Cryptosporidium parvum revealed by PCR-restriction fragment length polymorphism (RFLP) and RFLP-single-strand conformational polymorphism analyses.

A glycoprotein (Cpgp40/15)-encoding gene of Cryptosporidium parvum was analyzed to reveal intraspecies polymorphism within C. parvum isolates. Forty-one isolates were collected from different geographical origins (Japan, Italy, and Nepal) and hosts (humans, calves, and a goat). These isolates were characterized by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP-single-strand conformational polymorphism (RFLP-SSCP) analyses of the gene for Cpgp40/15. The sequence analysis indicated that there was DNA polymorphism between genotype I and II, as well as within genotype I, isolates. The DNA and amino acid sequence identities between genotypes I and II differed, depending on the isolates, ranging from 73.3 to 82.9% and 62.4 to 80.1%, respectively. Those among genotype I isolates differed, depending on the isolates, ranging from 69.0 to 85.4% and 54.8 to 79.2%, respectively. Because of the high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotypes Ia1, Ia2, Ib, and Ie. The isolates of genotype II could be subtyped as genotypes IIa, IIb, and IIc. The isolates from calves, a goat, and one Japanese human were identified as genotype II. Within genotype II, the isolates from Japan were identified as genotype IIa, those from calves in Italy were identified as genotype IIb, and the goat isolate was identified as genotype IIc. All of the genotype I isolates were from humans. The Japanese isolate (code no. HJ3) and all of the Nepalese isolates were identified as genotypes Ia1 and Ia2, respectively. The Italian isolates were identified as genotype Ib, and the Japanese isolate (code no. HJ2) was identified as genotype Ie. Thus, the PCR-RFLP-SSCP analysis of this glycoprotein Cpgp40/15 gene generated a high resolution that has not been achieved by previous methods of genotypic differentiation of C. parvum.

Amino Acid Sequence↗

PubMed2003

Age-related changes in peroxisomal membrane protein 70 and superoxide dismutase 1 in transgenic G93A mice.

Peroxisomal membrane protein 70 (PMP70) and Cu/Zn superoxide dismutase (SOD1) were examined in the spinal cords of transgenic (Tg) mice expressing a human mutant SOD1 protein (G93A) and their age-matched controls at 8, 20 and 32 weeks by immunohistochemistry. At pre-symptomatic 20 weeks and symptomatic 32 weeks, PMP70 was reduced in the cytoplasm of motor neurons in Tg animals and increased in glial cells in anterior horn at late age. SOD1 showed a progressive increase of dot-like deposits in the neuropil of anterior horn of Tg mice, and a late decrease of signal intensity in the white matter and motor neurons at 32 weeks. It is conceivable that reduction of PMP70 might underlie decrease in peroxisomal functions and increase in oxidative stress that is well documented in this animal model.

ATP-Binding Cassette Transporters↗

PubMed2003

Single-nucleotide polymorphisms in uncoding regions of ALS2 gene of Japanese patients with autosomal-recessive amyotrophic lateral sclerosis.

ALS2 is an autosomal recessive form of amyotrophic lateral sclerosis (AR-ALS) with juvenile onset, and has been mostly found in North African and Middle Eastern countries. Deletion mutations in the coding exons of a new gene ALS2, encoding a protein with guanine-nucleotide exchange factor (GEF) domains, have recently been identified in ALS2 patients. These mutations are predicted to cause a loss of protein function, indicating that ALS2 is the causative gene underlying ALS2. To examine whether ALS2 is mutated in Japanese ALS patients sharing some characteristics of ALS2, we analyzed ALS2 gene from three patients with AR-ALS. While no deletion mutation was detected in the coding regions of ALS2 gene, several single-nucleotide polymorphisms (SNPs) that have been found in healthy controls as well as in Tunisian ALS2 patients were found mostly in intronic regions of the gene. These results suggest that deletion mutations in ALS2 gene detected in ALS2 patients seem to be uncommon in Japanese AR-ALS, and that SNPs in uncoding regions might possibly be relevant to predisposition to ALS.

Adult↗

PubMed2003

Molecular cloning and characterization of a serine proteinase gene of Trichinella spiralis.

A serine proteinase gene was isolated from a cDNA library of Trichinella spiralis muscle larvae at 30 days post-infection (PI). The library was immunoscreened with T. spiralis-infected sera. A clone, designated Ts23-2, contained a cDNA transcript of 1,445 bp that encoded a putative signal peptide of 27 amino acids, a proregion of 20 amino acids, and a predicted mature enzyme of 374 amino acids. The predicted molecular mass of the Ts23-2 mature protein was 42.3 kDa. The enzyme comprised 2 regions, a catalytic domain of 234 residues and a C-terminal domain. The closest homologues of the Ts23-2 mature protein were serine proteinases from a wide range of organisms. The catalytic domain of the Ts23-2 clone was expressed as a proform in Escherichia coli. The recombinant protein cleaved serine proteinase-specific synthetic peptide substrates, and class-specific inhibitors of serine proteinases inhibited the enzymatic activity. Antibody against the Ts23-2 recombinant protein stained proteins migrating at about 51 and 33 kDa in crude extracts from 30-day PI muscle larvae and 18-day PI muscle larvae, but it failed to stain any proteins in crude extracts from newborn larvae and adult worms or in excretory-secretory products from 30-day PI muscle larvae. Production of the mRNA transcript for the Ts23-2 gene was mainly restricted to the 30-day PI muscle larvae, suggesting stage-specific expression. Intense staining with the anti-Ts23-2 serum was found within the parasites at the muscle stage of 30 days PI.

Amino Acid Sequence↗

PubMed2003

Molecular comparative studies among Blastocystis isolates obtained from humans and animals.

This study compared specific polymerase chain reaction (PCR) primers and phylogenetic tree analysis of restriction fragment length polymorphism using the small subunit ribosomal RNA gene in various Blastocystis populations. A phylogenetic tree was constructed using 12 restriction enzymes and a sample pool of 22 isolates, including 2 reference strains and Proteromonas lacertae as an outgroup. The analysis showed that the 22 isolates could be separated into 7 clusters. Four of the 7 clusters were mixed groups that comprised isolates from both humans and nonhuman hosts. The other 3 clusters contained isolates from humans or nonhuman hosts only. The phylogenetic analysis also showed that B. hominis isolates from geographical separated areas did not necessarily cluster in the genetically different groups. The results of genetic homology and phylogenetic tree analysis among Blastocystis isolates from humans and animals indicated that all isolates from animals appear to be B. hominis. Polymerase chain reaction amplifications using previously described and newly defined specific primers mirrored the clusters obtained by the phylogenetic tree analysis. Our results show that primer PCR can be used as a powerful tool for the typing of Blastocystis populations.

Animals↗