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Biomedical subjects

I Nagano

Publications and source records attributed to I Nagano.

At least 19 recordsLinked to original sources
PubMed2006

Molecular cloning and characterization of an Rcd1-like protein in excretory-secretory products of Trichinella pseudospiralis.

A cDNA library was constructed from muscle larvae of Trichinella pseudospiralis. A cDNA clone, designated as Tp8 contained a cDNA transcript of 1326 bp length with a single open reading frame, which encoded 303 amino acid residues (34,187 Da, estimated molecular mass). The predicted amino acid sequence of the clone had an identity of approximately 60% to the Rcd1 (Required cell differentiation 1) -like proteins among a wide range of organisms. Real-time quantitative polymerase chain reaction results showed that the transcription level of Tp8 gene reached the highest value in adult worms, and that the transcription level in muscle larvae before stichosome formation was higher than in muscle larvae after stichosome formation. The recombinant Tp8 protein migrated at 37 kDa and reacted to antibody against T. pseudospiralis excretory-secretory (E-S) products and sera from mice infected with T. pseudospiralis. An antibody against the Tp8 recombinant protein could stain proteins migrating at approximately 34 kDa (which is the expected size from the sequence) on Western blotting of E-S products from muscle larvae. An immunocytochemical study showed that the Tp8 protein was present within the stichocyte of muscle larvae and adults worms.

Amino Acid Sequence↗
PubMed2006

Involvement of the c-Ski oncoprotein in cell cycle arrest and transformation during nurse cell formation after Trichinella spiralis infection.

The role of c-Ski, an oncoprotein encoded by the oncogene, c-ski, in Trichinella spiralis-infected muscle tissues during nurse cell formation, was investigated by following the expression kinetics and distribution of c-Ski (both protein and mRNA) in the infected muscle cell, as well as the expression kinetics of the transforming growth factor beta (TGF-beta) signaling pathway factor genes (TGF-beta, Smad2 and Smad4) which cooperate with c-Ski. Immunohistochemical analysis using an anti-c-Ski antibody indicated that in the early stages of infection (13 and 18 days post-infection (p.i.)) the increased expression of the c-Ski protein was limited to the eosinophilic cytoplasm and not the enlarged nuclei or basophilic cytoplasm. At a later stage of infection (23 and 28 days p.i.) the c-Ski protein was limited to the enlarged nuclei in the basophilic cytoplasm, rather than the eosinophilic cytoplasm. At 48 days p.i., the c-Ski protein was barely detectable. Real-time PCR analysis showed that expression of the c-ski gene increased from 13 days p.i., reached a peak at 23-28 days p.i. and then decreased to a low level by 48 days p.i. Expression kinetics for the TGF-beta signaling pathway factor genes (TGF-beta, Smad2 and Smad4) were similar to that of c-ski. These findings provide evidence that the c-Ski protein is involved in nurse cell formation through the TGF-beta signaling pathway process in the host cell nucleus.

Animals↗
PubMed2005

Trichinella pseudospiralis infection is characterized by more continuous and diffuse myopathy than T. spiralis infection.

A time course study was performed to reveal the sequence of histopathology after Trichinella spiralis or T. pseudospiralis infection in mice. A cyst was formed in the former case by about 18 days post infection and prominent myopathy was restricted within the cyst. In the latter case, however, no typical cyst was formed, and myopathy spread diffusely over the infected muscle tissues occupying half the area of muscle sections. An electron microscope observation revealed that the disintegration of muscle cells was delayed in T. pseudospiralis infection than in T. spiralis infection. Quantitative reverse transcription polymerase chain reaction (RT-PCR) showed that apoptosis-related genes were expressed for a longer term in muscles infected with T. pseudospiralis than in those with T. spiralis, although the same spectrum of genes are mobilized. Examined apoptosis-related genes included tumor suppressor genes p53, p53; mouse double minute 2, MDM2; cyclin-dependent kinase inhibitor p21 (WAF1), p21(waf) ; Bcl-2 associated protein X, BAX; apoptotic protease activating factor 1, Apaf-1; Caspase 9 and serine/ threonine protein kinase, PKB. Micro-dissection of the infected muscle tissue and subsequent RT-PCR confirmed that the expressions of these genes are restricted to tissue with myopathy. Thus, the expression of the apoptosis-related genes correlated with continuous and diffuse myopathy caused by T. pseudospiralis infection.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2005

Molecular cloning and functional expression of enolase from Trichinella spiralis.

The cDNA library was constructed from muscle larvae of Trichinella spiralis, and immunoscreened with sera from mice infected with T. spiralis. A cDNA clone, designated as TsENO, encoded 2-phospho-D-glycerate hydro-lyase (enolase) that catalyzed a reversible conversion of 2-phospho-D-glyceric acid (2PGA) to phosphoenolpyruvate (PEP) in the glycolytic pathway. The recombinant TsENO protein was produced in an Escherichia coli expression system. The recombinant full-length TsENO protein had no activity in the conversion of 2PGA to PEP, but gained enolase activity after cutting off the signal peptide from the full-length protein. There was no meaningful difference in the expression level of TsENO gene at three distinct stages of T. spiralis. Also, antibody against the recombinant TsENO protein reacted with crude extract of muscle larvae, but not with the excretory and secretory products.

Amino Acid Sequence↗
PubMed2005

Reduction of a vascular endothelial growth factor receptor, fetal liver kinase-1, by antisense oligonucleotides induces motor neuron death in rat spinal cord exposed to hypoxia.

Vascular endothelial growth factor (VEGF) is reported to play a neuroprotective role through a VEGF receptor, fetal liver kinase-1 (Flk-1) in vitro. We investigated whether reduction of Flk-1 could induce motor neuron loss in rat spinal cord by inhibiting the expression of Flk-1 in rat spinal cord using antisense oligodeoxynucleotides (ODNs) against the Flk-1 receptor. Rat spinal cord was repetitively exposed to 12% hypoxia, and the change of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the mitogen-activated protein kinase kinase (MEK)/extracellular-signal-regulated kinase (ERK) pathway was examined. Intrathecal infusion of Flk-1 antisense ODNs for 7 days suppressed almost completely Flk-1 expression in the lumbar segment of the spinal cord and was followed by a hypoxic challenge with 12% oxygen for 1 h that was repeated for 7 more days. In the lumbar segment, we observed that reduced Flk-1 expression and hypoxic challenge for 7 days resulted in approximately 50% loss of motor neurons, in which the activation of Akt and ERK, that is, increased levels of phosphorylated-Akt and of phosphorylated-ERK by hypoxia, was markedly inhibited. In contrast, the reduction of Flk-1 expression alone did not induce motor neuron loss. These results suggest that VEGF exerts its protective effect on motor neurons against hypoxia-induced toxicity by the Flk-1 receptor through the PI3-K/Akt and the MEK/ERK signaling pathways.

Animals↗
PubMed2005

A spectrum of functional genes mobilized after Trichinella spiralis infection in skeletal muscle.

Trichinella spiralis infection causes the transformation of infected muscle cells, which leads to nurse cell formation. To search for the candidate genes responsible for nurse cell formation, cDNA microarray analysis of muscle tissues was performed before and after Trichinella infection. The Atlas mouse 1.2 cDNA expression microarray revealed the expression profiles of 1176 known genes. Out of these, 311 gene expressions were detected in normal and/or infected muscles. After the infection, 184 out of the 311 genes increased in expression by more than 3-fold. These included genes responsible for cell differentiation, proliferation, cell cycle and apoptosis. Thus this study suggested candidate genes for further investigation to dissect the molecular mechanisms of nurse cell formation.

Animals↗
PubMed2005

Tumor necrosis factor receptor-mediated apoptosis in Trichinella spiralis-infected muscle cells.

In order to reveal the mechanisms underlying nurse cell formation during Trichinella spiralis infection, the expression of the factors of tumor necrosis factor-alpha (TNF-alpha)/TNF receptor 1 (TNFR-1) signalling pathway mediating apoptosis was investigated. The analysed factors included TNF-alpha, TNFR-1, TNF receptor-associated death-domain (TRADD), caspase 3, caspase 8, TNF receptor associated factor-2 (TRAF2) and receptor interactive protein (RIP), all of which are involved in the TNF-alpha/TNFR-1 signalling pathway-mediated apoptosis. The quantitative RT-PCR indicated that the infected muscle tissues up-regulate the expression of pro-apoptosis genes (TNF-alpha, TNFR-1 and TRADD, caspase 3 and caspase 8), and anti-apoptosis genes (TRAF2 and RIP) at the beginning of cyst formation. The expression returned to the normal level after cyst formation. The quantitative RT-PCR analysis of mRNA from tissue samples isolated by laser capture micro-dissection confirmed that the up-regulation of these genes was restricted in infected muscle cells, was not in the inflammation cells around infected muscle cells nor in normal muscle cells. The in situ localization study of proapoptosis (TRADD, caspase 3) and anti-apoptosis gene products (TRAF2) indicated that these were expressed in the basophilic cytoplasm (infected muscle cell origin) of the nurse cells. Thus the present study suggests that the TNF-alpha/ TNFR-1 signalling pathway is involved in nurse cell formation.

Animals↗
PubMed2005

What is the role of p53 during the cyst formation of Trichinella spiralis ? A comparable study between knockout mice and wild type mice.

During the cyst formation of Trichinella spiralis, the infected muscle cell undergoes basophilic change and apoptosis, which results in nurse cell formation. This study revealed expression kinetics of some apoptosis genes such as p53 and its closely related genes (tumor suppressor genes p53, p53; mouse double minute 2, MDM2; cyclin-dependent kinase inhibitor p21, p21waf). RT-PCR (reverse transcription polymerase chain reaction) results showed that these genes were temporarily expressed in the infected muscles during the cyst formation period, but not in normal muscles (or very low if any), which suggested the involvement of these apoptosis genes in the nurse cell formation. Cysts and neighbouring muscle cells were separately collected and RT-PCR was performed, which suggested that p53 was expressed in the cysts. An immunocytochemical study showed that p53 was expressed in the nucleoplasm of basophilic cell in the cyst and Trichinella larvae, which suggested involvement of these apoptosis genes in the nurse cell formation. The same p53 expression kinetic study was performed on p53 knockout mice. The knockout mice did not express p53 genes, but expressed the other apoptosis genes in the same kinetics with only minor exceptions, suggesting that the expressions of these genes during the cyst formation were more or less p53-independent. There were no differences in the number and morphology of the cysts between the knockout mice and wild type mice. Thus apoptosis seen during the Trichinella cyst formation can be operated in the presence or absence of p53.

Animals↗
PubMed2004

Expression of apoptosis-related factors in muscles infected with Trichinella spiralis.

We found that the expression of mitochondrial apoptosis related genes (Bcl-2 associated protein X, BAX; apoptotic protease activating factor 1, Apaf-1; Caspase 9 and serine/threonine protein kinase, PKB) is elevated in Trichinella spiralis-infected muscles during encapsulation. Micro-dissection of the capsule and subsequent reverse transcription polymerase chain reaction (RT-PCR) confirmed that the expressions of these genes are restricted to the nurse cell. Immunocytochemistry revealed that pro-apoptosis factor (BAX, Apaf-1 and Caspase 9) are predominantly expressed in the basophilic cytoplasm (infected muscle cell origin) and anti-apoptosis factor (PKB) in the eosinophilic cytoplasm (satellite cell origin) of the nurse cell. Electron microscopy revealed that the pre-existing mitochondria in the muscle cells became swollen and disappeared immediately after newborn larva invasion, but new mitochondria of smaller size appeared in the cytoplasm. Nuclear fragmentation and condensation were observed in basophilic cytoplasm which is known to die. Together, the results suggest that the infected muscle cells transform but die through the process of apoptosis which is triggered by factors from the newly formed mitochondria. The anti-apoptosis factor may help the eosinophilic cytoplasm with its survival to ensure nurse cell function.

Animals↗
PubMed2004

Differences and similarities of nurse cells in cysts of Trichinella spiralis and T. pseudospiralis.

The nurse cell in the cyst of Trichinella spiralis comprises at least two kinds of cytoplasm, derived from muscle or satellite cells, as indicated by the pattern of staining using regular dye (haematoxylin and eosin, or toluidine blue), alkaline phosphatase (ALP) expression, acid phosphatase (ACP) expression and immunostaining with an anti-intermediate filament protein (desmin or keratin). Muscle cells undergo basophilic changes following a T. spiralis infection and transform to the nurse cells, accompanied by an increase in ACP activity and the disappearance of desmin. Satellite cells are activated, transformed and joined to the nurse cells but remain eosinophilic. The eosinophilic cytoplasm is accompanied by an increase in desmin and ALP expression but not an increase in ACP activity. Differences in the staining results for ALP or ACP suggest that the two kinds of cytoplasm have different functions. Trichinella pseudospiralis infection results in an increase of ACP activity at a later stage than T. spiralis. There is also a difference in the location pattern of ACP in the cyst of T. spiralis compared with T. pseudospiralis. In T. spiralis, ACP is diffused within the cell, but in T. pseudospiralis, ACP distribution is spotty corresponding to the location of the nucleus. Trichinella pseudospiralis infection is accompanied by a slight increase in ALP activity. Activated satellite cells following a T. pseudospiralis infection exhibit an increase in desmin expression. The present study therefore reveals that nurse cell cytoplasm differs between the two Trichinella species and between the two origins of cytoplasm in the cyst of T. spiralis.

Acid Phosphatase↗
PubMed2004

Severity dependent up-regulations of LOX-1 and MCP-1 in early sclerotic changes of common carotid arteries in spontaneously hypertensive rats.

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) and monocyte chemoattractant protein-1 (MCP-1) are molecules involving in the initiation and progression of atherosclerosis. In order to examine a possible difference in LOX-1 and MCP-1 expressions depending on the severity of early stage of atherosclerosis, we investigated atherosclerotic changes by exposure to hypertension and hyperlipidemia in common carotid arteries (CCAs) of stroke-prone spontaneously hypertensive rat (SHR-SP). Three rat model groups such as control [Wistar Kyoto rat (WKY) group], hypertension (SHR-SP group) and hypertension + hyperlipidemia [SHR-SP + high fat and cholesterol (HFC) group] were used. Body weights, brain weights, systolic blood pressures and serum levels of total cholesterol, low-density lipoprotein and triglyceride were measured at 0, 5, 10 and 15 days after appropriate diet. Immunohistochemistry showed that the positive area and the strength of LOX-1 and MCP-1 were larger in the SHR-SP + HFC group than in the SHR-SP group, while no immunoreactivities were found in the WKY group. Conventional RT-PCR and real-time PCR analyses showed that mRNAs of those in the SHR-SP group were higher with greater up-regulation in the SHR-SP + HFC group. LOX-1 and MCP-1 expressions were coordinately up-regulated at mRNA and protein levels in an early stage of sclerosis depending on the severity of atherosclerotic stress. Activations of LOX-1 and MCP-1 are collectively involved in the early stage of atherosclerosis.

Animals↗
PubMed2003

Highly polysialylated neural cell adhesion molecule (PSA-NCAM) positive cells are increased and change localization in rat hippocampus by exposure to repeated kindled seizures.

The highly polysialylated neural cell adhesion molecule (PSA-NCAM) is involved in migration of neural stem cells as well as in neural plasticity. Immunoreactive PSA-NCAM expression was examined in rats with repeated exposure to amygdaloid kindled generalized seizures (GS). The number of PSA-NCAM positive cells in the bilateral dentate gyrus (DG) increased significantly from GS. Although the total number of positive cells was not significantly different between animals with 3 times GS (3 GS) and 30 times GS (30 GS), in the latter group a greater number of positive cells was observed in the outer granule cell layer (GCL) and a marked extension of immunopositive dendrites to the molecular layer. These observations indicate that increased migration of newly generated cells as well as plastic changes of preexisting neural cells occur in response to recurrent GS. This may contribute to an abnormal reconstruction of the synaptic network in the hippocampus and, thus, epileptogenicity from kindling.

Animals↗
PubMed2003

Changes of free cholesterol and neutral lipids after transient focal brain ischemia in rats.

We investigated temporal and spatial changes of free cholesterol (FC) and neutral lipids (NLs) after brain ischemia with filipin complex staining to detect mainly FC and Nile Red staining for NLs such as cholesteryl ester (CE) and triacylglyceride (TAG). Filipin stainining decreased during 1 to 7 d and lost at 21 d after transient middle cerebral artery (MCA) occlusion in the ischemic core, but did not change in the penumbra. Nile Red positive droplets reached the maximum at 7 d after transient MCA occlusion (MCAO) and gradually decreased in the core, while the peak time delayed in the penumbra. Most Nile Red positive droplets were double positive for ED1 in the core, and were localized within GFAP positive cells in the penumbra. The present study suggests that changes of FC and NLs are different temporally and spatially between the ischemic core and the penumbra in relation to degenerative and regenerative neural processes following brain ischemia. Macrophages and astrocytes are presumed to play important roles in lipid metabolism for neural reorganization of the ischemic brain injury in the ischemic core and the penumbra, respectively.

Animals↗
PubMed2003

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

Oligodendrocyte progenitor cells (OPCs) mature to oligodendrocyte and remyelinate axons. OPCs express neuron-glial antigen 2 (NG2) chondroitin sulfate proteoglycan. We stained NG2 to investigate the effect of aging on the OPCs after cerebral ischemia. NG2 positive cells were examined at 1, 3, 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. At 7 day, 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 more highly branched in the young than the aged brains. These results suggest that the young brain shows a higher potential of proliferation and process branching of OPCs than the aged brain.

Aging↗