Biomedical subjects

H Hatanaka

Publications and source records attributed to H Hatanaka.

At least 55 records · Page 3Linked to original sources

PubMed2000

Involvement of cystatin C in oxidative stress-induced apoptosis of cultured rat CNS neurons.

Oxidative stress is involved in neuronal degeneration in cerebrovascular injury, neuropathology and aging. When rat CNS neurons were cultured in a high (50%) oxygen atmosphere, the neurons died. This high oxygen-induced cell death showed features of apoptotic cell death, characterized by DNA fragmentation, and was blocked by inhibitor of protein synthesis. We found that cystatin C and HuC mRNA, the products of which are an inhibitor of cysteine proteases and an RNA binding protein, respectively, were up-regulated in neurons cultured in the high oxygen atmosphere. In the present study, we focused on cystatin C. Cystatin C protein levels were also increased in neurons cultured in the high oxygen atmosphere. In situ hybridization with an RNA probe for rat cystatin C and immunocytochemistry with anti-human cystatin C antibody showed that microtubule-associated protein 2 (MAP2)-positive neurons expressed cystatin C mRNA and protein, respectively, in the high oxygen atmosphere. These results indicated that oxidative stress stimulates an increase in cystatin C expression in cultured neurons, and that cystatin C might have important roles in regulation of apoptosis elicited by oxidative stress.

Animals↗

PubMed2000

SNRK, a member of the SNF1 family, is related to low K(+)-induced apoptosis of cultured rat cerebellar granule neurons.

When cerebellar granule neurons obtained from 11-day-old rats were cultured first in high K(+) medium for 4 days, followed by culture in low K(+) medium, the neurons underwent apoptosis and died. This cell death was prevented by actinomycin D, an inhibitor of RNA synthesis. Commitment time of the protective effect of RNA synthesis inhibition on the cell death was examined by adding actinomycin D at various time points after the switch to the low K(+) medium. More than 50% of the cells died when actinomycin D was added 3 h after changing to the low K(+) medium. To identify what kinds of newly synthesized genes are involved in regulation of the low K(+)-induced death, we performed PCR-based differential subtraction analysis using RNA prepared from the cultured neurons 0 and 3 h after changing to low K(+) medium. We isolated a clone that showed an increase in its mRNA level after changing to the low K(+) medium. This clone encoded the 3' untranslated region of SNRK, a serine/threonine kinase. Tissue distribution analysis showed that the mRNA was expressed mainly in the brain and testis. Developmental analysis in the brain showed that the mRNA expression increased in an age-dependent manner until P28, and was slightly decreased in adults. In situ hybridization analysis showed that the mRNA was expressed throughout the brain. The mRNA was shown to be expressed in neurons by double staining with anti-MAP2 antibody. In addition, anti-N-terminal SNRK antibody stained the nuclei of cultured rat cerebellar granule neurons. These results suggested that SNRK may be involved in regulation of low K(+)-induced apoptosis of cultured cerebellar granule neurons.

Animals↗

PubMed2000

Role of p53 in DNA strand break-induced apoptosis in organotypic slice culture from the mouse cerebellum.

Apoptosis occurs not only in mitotic cells but also in postmitotic neuronal cells. We previously suggested that the tumor suppressor gene p53 is required for DNA strand break-induced apoptosis in dissociated culture of cerebellar granule neurons. In this study, we examined the role of p53 in apoptosis using organotypic slice culture of cerebellum from p53 null and wild-type mice. Exposure to bleomycin significantly increased the numbers of TUNEL-, p53-, and c-Jun-positive neurons in the wild-type mouse cerebellar internal granular layer (IGL) and Purkinje cell layer (PL). However, in p53-deficient mice, these responses were not observed. These results are consistent with our previous observations in dissociated neuronal culture showing that the amount of c-Jun protein increases significantly after addition of bleomycin in p53 wild-type cerebellar granule cells. The results presented here also indicate that p53 is involved in DNA strand break-induced apoptosis of fully postmitotic central nervous system neurons and suggest that c-Jun expression occurs downstream of p53 expression.

Animals↗

PubMed2000

Chronic fentanyl treatments induce the up-regulation of mu opioid receptor mRNA in rat pheochromocytoma cells.

Chronic activation of adenylate cyclase-cAMP-cAMP-dependent protein kinase (PKA) systems by administration of opioid receptor agonists has been considered as one of the mechanisms of opioid tolerance and dependence. Although analysis of the micro opioid receptor (MOR) gene suggests that cAMP-related signal transduction systems regulate the expression of this gene, which transcription factors affect the MOR gene expression in brain and neural cells has not been clarified. This study deals with the effects of fentanyl on MOR mRNA levels in the rat pheochromocytoma cell line (PC12 cells). PC12 cells were cultured in medium with clinically relevant concentrations of fentanyl. The quantitative reverse transcription and polymerase chain reaction (RT-PCR) method was used for determination of MOR mRNA. Treatment of PC12 cells with fentanyl induced the MOR mRNA up-regulation in a concentration- and time-dependent manner. A cAMP analogue also up-regulated MOR mRNA. The intracellular cAMP level increased after fentanyl treatment. A PKA inhibitor blocked the MOR mRNA up-regulation by fentanyl and the cAMP analogue. Expression of a dominant inhibitory Ras also inhibited the MOR mRNA up-regulation. Fentanyl-induced up-regulation of MOR mRNA via activation of cAMP signaling may be important in compensating for the MOR reduction during long-term treatment of PC12 cells with fentanyl. The present study could be relevant to understanding the molecular mechanisms of opioids in a state of drug tolerance or dependence, and in patients under anesthesia or being treated for pain.

Analgesics, Opioid↗

PubMed2000

BDNF rapidly induces aspartate release from cultured CNS neurons.

We investigated the effects of brain-derived neurotrophic factor (BDNF) on aspartate release from cultured cerebellar neurons. This release occurred within 1 min after the addition of 100 ng/ml BDNF. The amount of aspartate released was less than that of glutamate. Aspartate release induced by BDNF was rapid and transient, as in the case of glutamate. Although high potassium evoked the release of both excitatory (glutamate and aspartate) and inhibitory (GABA and glycine) amino acid transmitters, BDNF only induced glutamate and aspartate release. BDNF-induced aspartate release was completely blocked by pretreatment with K252a or TrkB-IgG. The aspartate release induced by BDNF was not dependent on the extracellular Ca(2+), but required intracellular Ca(2+) mobilization. These results showed that BDNF may be involved in excitatory transmission using aspartate as well as glutamate through TrkB-mediated signaling in cerebellum.

Animals↗

PubMed2000

Clinical implications of interleukin (IL)-10 induced by non-small-cell lung cancer.

BACKGROUND: The type 2 cytokine interleukin (IL)-10 has been reported to inhibit the antitumour activity of the regional immunity against various neoplasms. Certain lung cancers produce IL-10, but the clinical significance of IL-10 expression is not well understood. PATIENTS AND METHODS: We examined IL-10 and IL-10 receptor (IL-10R) mRNA expression in 82 non-small-cell lung cancers (NSCLC) by reverse transcription-polymerase chain reaction (RT-PCR) assay. Immunohistochemistry (IHC) and enzyme immunoassay (EIA) were applied to evaluate the cellular localisation and the serum levels of IL-10. RESULTS: RT-PCR assay revealed IL-10 mRNA expression in 68 (83%) of 82 NSCLC surgical specimens (40 of 50 adenocarcinomas, 22 of 26 squamous cell carcinomas, 5 of 5 large-cell carcinomas, 1 of 1 adenosquamous-cell carcinoma). RT-PCR assay also revealed IL-10R mRNA expression in 79 cases of NSCLC (96.1%). IL-10 expression was confirmed within tumour cells by IHC. EIA showed no significant serum IL-10 elevation in the 12 NSCLC positive for IL-10 mRNA expression (0-2.99 pg/ml). The NSCLC patients with IL-10 production showed significantly poorer prognosis than those without IL-10 production (P < 0.05, Kaplan Meier, log-rank test). CONCLUSIONS: These results suggested that the cytoplasmic IL-10 correlated to clinical prognosis, and that IL-10 expression is a prognostic factor for NSCLC.

Carcinoma, Non-Small-Cell Lung↗

PubMed2000

Effects of proline mutations in the major house dust mite allergen Der f 2 on IgE-binding and histamine-releasing activity.

Der f 2 is the major group 2 allergen from house dust mite Dermatophagoides farinae and is composed of 129 amino-acid residues. Wild-type and six proline mutants of Der f 2 (P26A, P34A, P66A, P79A, P95A, and P99A) expressed in Escherichia coli were refolded and purified. Formations of intramolecular disulfide bonds in the purified proteins were confirmed correct. The apparent molecular masses analyzed by gel-filtration were 14-15 kDa. The IgE-binding capacity in the sera of seven mite-allergic patients, inhibitory activity for IgE-binding to immobilized wild-type Der f 2, and activity to stimulate peripheral blood basophils to release histamine in two volunteers were analyzed. P95A and P99A, which slightly differed from the wild-type Der f 2 in their CD spectrum, showed reduced IgE-binding, reduced inhibitory activity, and less histamine-releasing activity than the wild-type. P34A also showed reduced allergenicity. Considering that Pro95, Pro99 and Pro34 are closely located in loops at one end of the tertiary structure of Der f 2, we concluded that these loop regions included an IgE-binding site common to all tested patients. P66A showed reduced IgE-binding in two sera out of seven. P26A and P79A showed no reduced allergenicity. However, in immunoblot analysis after SDS/PAGE under reduced conditions, P79A showed no or markedly reduced IgE-binding while the other mutants showed IgE-binding corresponding to that in the assay using correctly refolded proteins. This suggests that Pro79 is involved in refolding of Der f 2. The findings in this study are important for the understanding of the antigenic structure of mite group 2 allergens and for manipulation of the allergens for specific immunotherapy.

Allergens↗

PubMed2000

Pituitary adenylate cyclase-activating polypeptide promotes the survival of basal forebrain cholinergic neurons in vitro and in vivo: comparison with effects of nerve growth factor.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasointestinal polypeptide gene family for which neurotrophic activity has been postulated. PACAP mRNA is expressed in the developing and adult hippocampus, which is the principal target region of septal cholinergic neurons. We therefore studied the effects of PACAP on septal cholinergic neurons. In primary cultures from septum of embryonic and postnatal rats, PACAP increased the number of neurons immunohistochemically stained for the low-affinity nerve growth factor (NGF) receptor p75 and for the enzyme choline acetyltransferase (ChAT). PACAP also caused a corresponding increase in ChAT activity. In comparison, NGF had a greater effect than PACAP on the number of p75- and ChAT-positive neurons in these cultures. In vivo, following fimbria fornix transection, the number of immunohistochemically stained septal cholinergic neurons fell significantly to 18% in rats given continuous intracerebroventricular infusion of vehicle, whereas in rats given NGF the number of these neurons did not differ significantly from unoperated controls. In PACAP-treated rats the number was 48% of unoperated values, which represented a significant increase compared with vehicle-treated rats and a significant decrease compared with NGF-treated rats or unoperated controls. Double-staining experiments revealed that most ChAT-positive neurons in rat medial septum also express PACAP receptor 1. Together the results show that PACAP promotes the survival of septal cholinergic neurons in vitro, and after injury in vivo, suggesting that PACAP acts as a neurotrophic factor influencing the development and maintenance of these neurons.

Acetylcholine↗

PubMed2000

Regulation of alpha3 nicotinic acetylcholine receptor subunit mRNA levels by nerve growth factor and cyclic AMP in PC12 cells.

To investigate the effects of nerve growth factor (NGF) and cyclic AMP (cAMP) on the level of the nicotinic acetylcholine receptor subunit alpha3 mRNA, we used PC12h cells, PC12 cells expressing dominant-negative Ras protein, and the parental PC12 cells. PC12h cells have NGF-responsive tyrosine hydroxylase activity. Expression of dominant-negative Ras protein prevents the signaling through the Ras-mitogen-activated protein kinase cascade. The morphological changes of the parental PC12 cells in response to NGF and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPTcAMP), a cell-penetrating cAMP analogue, were similar to those of PC12h cells. NGF up-regulated the alpha3 mRNA level in PC12h cells and down-regulated the alpha3 mRNA level in the parental PC12 cells. Expression of dominant-negative Ras protein and an inhibitor of mitogen-activated protein kinase kinase inhibited the effects of NGF on alpha3 mRNA level. CPTcAMP down-regulated the alpha3 mRNA level in all three PC12 cell lines. An inhibitor of protein kinase A inhibited the CPTcAMP-induced down-regulation of alpha3 mRNA. The alpha3 mRNA down-regulation required prolonged treatment with CPTcAMP even after cAMP response element binding protein phosphorylation was decreased. Membrane depolarization with high K+ had no effect on the alpha3 mRNA level in PC12h cells. Based on these results, we propose that at least two unknown effectors regulate alpha3 mRNA levels in PC12 cells.

Animals↗

PubMed2000

Brain-derived neurotrophic factor accelerates nitric oxide donor-induced apoptosis of cultured cortical neurons.

Brain-derived neurotrophic factor (BDNF) is known to have important functions in neuronal survival, differentiation, and plasticity. In addition to its role as a survival-promoting factor, BDNF reportedly can enhance neuronal cell death in some cases, for example, the death caused by excitotoxicity or glucose deprivation. The cellular mechanism of the death-enhancing effect of BDNF remains unknown, in contrast to that of its survival-promoting effect. In this work, we found that BDNF markedly accelerated the nitric oxide (NO) donor-induced death of cultured embryonic cortical neurons. BDNF increased the number of cells with nuclear condensation and DNA fragmentation 24 h after treatment with the NO donor, but it did not change the number of those cells 36 h after the treatment. The BDNF-accelerated death of cortical neurons was inhibited by the addition of actinomycin D or cycloheximide. These results suggest that BDNF can accelerate apoptotic cell death elicited by NO donor. TrkB-IgG and K252a blocked the BDNF-induced acceleration of the death, indicating that the death-accelerating effect by BDNF is mediated by TrkB. In addition, the BDNF-accelerated apoptosis was inhibited by the addition of SB202190 and SB203580, specific inhibitors of p38 mitogen-activated protein kinase (MAPK), and U0126, a specific inhibitor of MAPK/ERK kinase 1, indicating that the activation of both p38 MAPK and ERK is involved in the signaling cascade of the BDNF-accelerated, NO donor-induced apoptosis.

Animals↗

PubMed2000

Facilitatory roles of novel compounds designed from cyclopentenone prostaglandins on neurite outgrowth-promoting activities of nerve growth factor.

Cyclopentenone prostaglandins (PGs) are known to arrest the cell cycle at the G(1) phase in vitro and to suppress tumor growth in vivo. However, their effects on neurons are unclear. Here, we report that some cyclopentenone PGs function as neurite outgrowth-promoting factors. They promoted neurite outgrowth from PC12 cells and from dorsal root ganglion explants but only in the presence of nerve growth factor (NGF). We refer to these PGs as neurite outgrowth-promoting PGs (NEPPs). Through study of the structure-function relationship of NEPP1-10 and related compounds, we found that the cross-conjugated dienone moiety of NEPPs was essential for promoting neurite outgrowth, and NEPP10 was concluded to be the best candidate for drug development. We also investigated the intracellular mechanism of the promotion by NEPPs and obtained evidence that immunoglobulin heavy chain binding protein/glucose-regulated protein 78 (BiP/GRP78) plays a role in the promotion, based on the following observations: Antisense nucleotides for BiP/GRP78 gene blocked the promotion of neurite outgrowth; BiP/GRP78 protein level increased in response to NEPPs; and overexpression of BiP/GRP78 protein by adenoviral gene transfer promoted the neurite outgrowth by NGF.

Adenoviridae↗

PubMed2000

BIT/SHPS-1 enhances brain-derived neurotrophic factor-promoted neuronal survival in cultured cerebral cortical neurons.

Brain-derived neurotrophic factor (BDNF) activates a variety of signaling molecules to exert various functions in the nervous system, including neuronal differentiation, survival, and regulation of synaptic plasticity. Previously, we have suggested that BIT/SHPS-1 (brain immunoglobulin-like molecule with tyrosine-based activation motifs/SHP substrate 1) is a substrate of Shp-2 and is involved in BDNF signaling in cultured cerebral cortical neurons. To elucidate the biological function of BIT/SHPS-1 in cultured cerebral cortical neurons in connection with its role in BDNF signaling, we generated recombinant adenovirus vectors expressing the wild type of rat BIT/SHPS-1 and its 4F mutant in which all tyrosine residues in the cytoplasmic domain of BIT/SHPS-1 were replaced with phenylalanine. Overexpression of wild-type BIT/SHPS-1, but not the 4F mutant, in cultured cerebral cortical neurons induced tyrosine phosphorylation of BIT/SHPS-1 itself and an association of Shp-2 with BIT/SHPS-1 even without addition of BDNF. We found that BDNF-promoted survival of cultured cerebral cortical neurons was enhanced by expression of the wild type and also 4F mutant, indicating that this enhancement by BIT/SHPS-1 does not depend on its tyrosine phosphorylation. BDNF-induced activation of mitogen-activated protein kinase was not altered by the expression of these proteins. In contrast, BDNF-induced activation of Akt was enhanced in neurons expressing wild-type or 4F mutant BIT/SHPS-1. In addition, LY294002, a specific inhibitor of phosphatidylinositol 3-kinase, blocked the enhancement of BDNF-promoted neuronal survival in both neurons expressing wild-type and 4F mutant BIT/SHPS-1. These results indicate that BIT/SHPS-1 contributes to BDNF-promoted survival of cultured cerebral cortical neurons, and that its effect depends on the phosphatidylinositol 3-kinase-Akt pathway. Our results suggest that a novel action of BIT/SHPS-1 does not occur through tyrosine phosphorylation of BIT/SHPS-1 in cultured cerebral cortical neurons.

Adenoviridae↗

PubMed2000

Shp-2 specifically regulates several tyrosine-phosphorylated proteins in brain-derived neurotrophic factor signaling in cultured cerebral cortical neurons.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophins, promotes differentiation and survival and regulates plasticity of various types of neurons. BDNF binds to TrkB, a receptor tyrosine kinase, which results in the activation of a variety of signaling molecules to exert the various functions of BDNF. Shp-2, a Src homology 2 domain-containing cytoplasmic tyrosine phosphatase, is involved in neurotrophin signaling in PC12 cells and cultured cerebral cortical neurons. To examine the roles of Shp-2 in BDNF signaling in cultured rat cerebral cortical neurons, the wild-type and phosphatase-inactive mutant (C/S mutant) forms of Shp-2 were ectopically expressed in cultured neurons using recombinant adenovirus vectors. We found that several proteins tyrosine-phosphorylated in response to BDNF showed enhanced levels of tyrosine phosphorylation in cultured neurons infected with C/S mutant adenovirus in comparison with those infected with the wild-type Shp-2 adenovirus. In addition, in immunoprecipitates with anti-Shp-2 antibody, we also observed at least four proteins that displayed enhanced phosphorylation in response to BDNF in cultured neurons infected with the C/S mutant adenovirus. We found that the Shp-2-binding protein, brain immunoglobulin-like molecule with tyrosine-based activation motifs (BIT), was strongly tyrosine-phosphorylated in response to BDNF in cultured neurons expressing the C/S mutant of Shp-2. In contrast, the level of BDNF-induced phosphorylation of mitogen-activated protein kinase and coprecipitated proteins with anti-Trk and Grb2 antibodies did not show any difference between neurons infected with these two types of Shp-2. Furthermore, the survival effect of BDNF was enhanced by the wild type of Shp-2, although it was not influenced by the C/S mutant of Shp-2. These results indicated that in cultured cerebral cortical neurons Shp-2 is specifically involved in the regulation of several tyrosine-phosphorylated proteins, including BIT, in the BDNF signaling pathway. In addition, the phosphatase Shp-2 may not influence the level of BDNF-induced activation of mitogen-activated protein kinase in cultured cortical neurons. Further, Shp-2 may have potential to positively regulate BDNF-promoting neuronal survival.

Adaptor Proteins, Signal Transducing↗

PubMed2000

An in vivo study of locking and grasping techniques using a passive mobilization protocol in experimental animals.

An in vivo experimental study was performed on the flexor profundus tendons of adult canines to determine tensile strength differences between locking and grasping technique repairs and to document the tensile strength using a passive mobilization protocol through the first 21 days following surgery. Forty experimental third and fourth flexor tendons of the right forepaws were lacerated and repaired with either of the 2 techniques. The experimental tendons were harvested at days 3, 7, 14, and 21 and were biomechanically tested. The third and fourth profundus tendons of the left forepaws were used as control (day 0) tendons; they were lacerated and repaired with the locking or grasping techniques at the time of harvest and also were biomechanically tested. The locking technique statistically improved tensile strength values at days 0, 3, and 21. The tensile strength decrease was minimal when using a passive mobilization protocol after surgery.

Analysis of Variance↗

PubMed2000

The mechanical properties of locking and grasping suture loop configurations in four-strand core suture techniques.

We have compared the effect of locking and grasping suture loop configurations in four-strand core suture techniques for tendon repair. Forty canine flexor digitorum profundus tendons were repaired with one of four suture techniques (the grasping cruciate, the double-modified grasping Kessler, the locking cruciate and the double-modified locking Kessler) and tested to failure in a tensile testing machine. The mode of failure in all the locking suture specimens was breakage of the sutures in the locking loops or at suture knots. The sutures did not pull out of the tendon, as was seen in the grasping suture specimens. The greatest tensile strength was found with the double-modified locking Kessler technique which incorporated eight rectangular locking loop configurations.

Animals↗

PubMed2000

Backbone dynamics of the c-Myb DNA-binding domain complexed with a specific DNA.

The DNA-binding domain of c-Myb consists of three imperfect tandem repeats, R1, R2, and R3. Each repeat contains three helices. The minimal DNA-binding domain is an R2R3 fragment. Here, we have examined the backbone dynamics of R2R3 in its DNA-bound form by NMR. Upon binding to DNA, the N- and C-termini, and the linker between R2 and R3 become less flexible. In the free form the third helix of R2 exhibits slow conformational exchange fluctuations owing to a cavity in the hydrophobic core of R2. Upon binding to DNA, the conformational exchange contributions in R2 are reduced but remain significant in NMR relaxation measurements. Upon binding to DNA, the third helix of R3 comes to exhibit significant chemical exchange contributions. These findings suggest that the orientations of the third helices of both R2 and R3 as to DNA are being chemically exchanged. In the DNA-bound form both R2 and R3 exhibit similar dynamical characters, except for amino acids Trp 95, Thr 96, and Val 103 of R2, which are located around the cavity of the unbound form. Upon binding to DNA, since Trp 95 moves into the cavity to fill it up, the local conformational exchange contributions seem to be still observable around the filled cavity.

Amino Acid Sequence↗

PubMed2000

Effect of suture size on locking and grasping flexor tendon repair techniques.

An experimental study was performed using human cadaver flexor tendons to investigate the effect of locking and grasping loop techniques on the tensile properties of repaired flexor tendons, which closely resemble the clinical model. Statistically significant improvement was observed only with the locking loop technique for ultimate and gap strength values using 2-0 core suture and ultimate strength values using 3-0 core suture. There was no statistically significant increase in tensile strength values using 4-0 core suture material. A heavier core suture used with the locking loop technique provided greater ultimate and gap strength of a repaired tendon than when used with the grasping loop technique.

Humans↗

PubMed2000

Vascularization is decreased in pulmonary adenocarcinoma expressing brain-specific angiogenesis inhibitor 1 (BAI1).

Brain-specific angiogenesis inhibitor 1 (BAI1) is a novel angioinhibitory factor. We examined BAI1 gene expression in 48 pulmonary adenocarcinoma specimens by RT-PCR. Vascular density (number and measurement area) was immunohistochemically evaluated in tumor specimens using anti-CD34 monoclonal antibody. BAI1 gene expression was detected in 38 of 48 pulmonary adenocarcinomas (79. 2%). Vascular number and measurement area were significantly decreased in the BAI1-positive pulmonary adenocarcinomas (19.3+/-4. 4/microm2 and 1.7+/-0.6%) as compared with those in the BAI1-negative carcinomas (75.5+/-42.7/microm2 and 5.5+/-1.5%) (Mann-Whitney U-test, p=0.0017). These results suggest that BAI1 expression may inhibit stromal vascularization in pulmonary adenocarcinoma.

Adaptor Proteins, Signal Transducing↗