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Biomedical subjects

K Kuwabara

Publications and source records attributed to K Kuwabara.

At least 37 records · Page 2Linked to original sources
PubMed2000

An unusual case of mediastinal lymphadenopathy caused by amyloidosis.

A 71-year-old Japanese female Nagasaki-atomic-bomb survivor was admitted for evaluation of a mediastinal mass. She was infected with human T-cell leukemia virus type I. Histological examination of the biopsy specimen, obtained thoracoscopically from the mass, revealed amyloid lymphadenopathy confirmed by Congo-red staining and electron-microscopic examination. Amyloid deposits and the serum monoclonal peak consisted of immunoglobulin lambda light chains. No atypical cells were detected in bone marrow. The case was diagnosed as an unusual amyloidosis affecting the mediastinal lymph node. No other sites were found to be affected by amyloidosis, although systemic involvement could not be conclusively ruled out.

Aged↗
PubMed1999

Deficiency of intercellular adhesion molecule 1 fails to mitigate selective neuronal death after transient global ischemia.

Recent studies have shown a crucial role of intercellular adhesion molecule 1 (ICAM-1) in expansion of infarction after focal cerebral ischemia. The purpose of the present study was to assess whether ICAM-1 is involved in selective neuronal vulnerability and reactive gliosis after transient forebrain ischemia. ICAM-1 knockout mice and wild-type mice were subjected to transient forebrain ischemia for 5, 10 or 15 min, and the hippocampus and caudoputamen were examined 7 days later with conventional histological and immunohistochemical methods. Bilateral common carotid artery occlusion with less than 10% of baseline cortical microperfusion for 10 or 15 min resulted in ischemic neuronal damage in the hippocampus and caudoputamen. The frequency and the severity of neuronal damage were similar in wild-type and knockout mice. Proliferation of reactive astrocytes in the hippocampus was also similar in both types of mice. Therefore, it is highly unlikely that ICAM-1 plays a key role in delayed neuronal death after transient global ischemia or in astroglial responses after ischemic neuronal injury.

Animals↗
PubMed1999

150-kDa oxygen-regulated protein (ORP150) suppresses hypoxia-induced apoptotic cell death.

To determine the contribution of 150-kDa oxygen-regulated protein (ORP150) to cellular processes underlying adaptation to hypoxia, a cell line stably transfected to overexpress ORP150 antisense RNA was created. In human embryonic kidney (HEK) cells stably overexpressing ORP150 antisense RNA, ORP150 antigen and transcripts were suppressed to low levels in normoxia and hypoxia, whereas wild-type cells showed induction of ORP150 with oxygen deprivation. Inhibition of ORP150 in antisense transfectants was selective, as hypoxia-mediated enhancement of glucose-regulated protein (GRP) 78 and GRP94 was maintained. However, antisense ORP150 transfectants displayed reduced viability when subjected to hypoxia, compared with wild-type and sense-transfected HEK cells. In contrast, diminished levels of ORP150 had no effect on cytotoxicity induced by other stimuli, including oxygen-free radicals and sodium arsenate. Although cellular ATP content was similar in hypoxia, compared with ORP150 antisense transfectants and wild-type HEK cells, suppression of ORP150 expression was associated with accelerated apoptosis. Hypoxia-mediated cell death in antisense HEK transfectants did not cause an increase in caspase activity or in cytoplasmic cytochrome c antigen. A well recognized inducer of apoptosis in HEK cells, staurosporine, caused increased caspase activity and cytoplasmic cytochrome c levels in both wild-type and antisense cells. These data indicate that ORP150 has an important cytoprotective role in hypoxia-induced cellular perturbation and that ORP150-associated inhibition of apoptosis may involve mechanisms distinct from those triggered by other apoptotic stimuli.

Apoptosis↗
PubMed1999

Species differences in fodrin proteolysis in the ischemic brain.

There has been growing evidence that the breakdown of cytoskeletal proteins is an important biochemical change leading to ischemic neuronal death. In the present study, we investigated species differences in the susceptibility of fodrin to calpain activation induced by cerebral ischemia in gerbils, rats, and mice. In vivo fodrin proteolysis and degradation of microtubule-associated protein 2 after complete ischemia occurred more rapidly in the hippocampus and cerebral cortex of the gerbil brain than in the corresponding area of the rat and mouse brain. The N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 injected intraperitoneally before ischemia did not diminish fodrin degradation in the gerbil hippocampus. In vivo fodrin proteolysis was inhibited at 33 degrees C and enhanced at 41 degrees C compared with proteolysis at 37 degrees C during ischemia. However, in vitro fodrin proteolysis after addition of Ca2+ into the crude membrane fraction did not show any differences among three species. Although it is highly unlikely that the difference in the sensitivity of NMDA receptor or the sensitivity of calpain activation to calcium was the crucial determinant of susceptibility of fodrin degradation in the gerbil brain, the present study clearly demonstrated that fodrin in the gerbil brain was more susceptible to calpain activation induced by ischemia than that in the rat and mouse brains. Enhanced proteolysis may be one of the reasons neurons in the gerbil brain are highly vulnerable to ischemia.

Animals↗
PubMed1999

Localization of curved DNA and its association with nucleosome phasing in the promoter region of the human estrogen receptor alpha gene.

We determined DNA bend sites in the promoter region of the human estrogen receptor (ER) gene by the circular permutation assay. A total of five sites (ERB-4 to -1, and ERB+1) mapped in the 3 kb region showed an average distance of 688 bp. Most of the sites were accompanied by short poly(dA) x poly(dT) tracts including the potential bend core sequence A2N8A2N8A2 (A/A/A). Fine mapping of the ERB-2 site indicated that this A/A/A and the 20 bp immediate flanking sequence containing one half of the estrogen response element were the sites of DNA curvature. All of the experimentally mapped bend sites corresponded to the positions of DNA curvature as well as to nucleosomes predicted by computer analysis. In vitro nucleosome mapping at ERB-2 revealed that the bend center was located 10-30 bp from the experimental and predicted nucleosome dyad axes.

Base Sequence↗
PubMed1999

Clinical and immunological significance of neopterin measurement in cerebrospinal fluid in patients with febrile convulsions.

Neopterin is synthesized mainly by monocytes/macrophages and is considered to be a marker for activation of the cellular immune system. It has been reported that cerebrospinal fluid (CSF) neopterin levels are significantly higher in patients with bacterial meningitis than in those with aseptic meningitis or non-pleocytotic CSF. In this study levels of neopterin and interferon-gamma (IFN-gamma) were measured in children with non-pleocytotic CSF. The CSF neopterin levels were significantly higher in patients with typical febrile convulsions (FCs) (15.0 +/- 4.5 nmol/l) than in those with pyrexia without convulsions (6.5 +/- 2.7 nmol/l) or convulsions without pyrexia, namely, epilepsy (4.8 +/- 2.4 nmol/l). The CSF neopterin/serum neopterin ratio (C/S ratio) was also higher in patients with typical FCs (1.54 +/- 0.83) than in those with pyrexia without convulsions (0.32 +/- 0.18) or convulsions without pyrexia (0.77 +/- 0.28). Patients with prolonged FCs tended to have higher CSF neopterin levels than those with typical FCs. There was also a tendency for CSF IFN-gamma levels to be higher in patients with FCs than in those with pyrexia without convulsions or convulsions without pyrexia. The results of the present study suggest that some immune activation in the central nervous system (CNS) compartment may be related to the mechanisms of FCs.

Child, Preschool↗
PubMed1999

Molecular cloning of a novel member of the HSP110 family of genes, ischemia-responsive protein 94 kDa (irp94), expressed in rat brain after transient forebrain ischemia.

To identify genes induced by transient forebrain ischemia, we used the mRNA differential display technique in the four-vessel occlusion model in rats. Some genes were identified as candidates that encode ischemia-responsive protein, and one of them was cloned as ischemia-responsive protein 94 kDa (irp94) from the rat hippocampal cDNA library. Sequence analysis suggested that rat irp94 was a transcriptional variant or a homologue of mouse apg-2 and human heat shock protein (hsp) 70RY and a member of the HSP110 family, because IRP94 was >90% identical to APG-2 and HSP70RY and approximately 60% identical to the other members of the HSP110 family. Although irp94 mRNA was constitutively expressed in the normal hippocampus, it was clearly enhanced 4-24 h after ischemia for 10 (1.9-fold increase) and 15 min (3.4-fold increase). These changes mainly occurred in neuronal cells, as judged by the localization of irp94 mRNA using in situ hybridization histochemistry. On the other hand, hyperthermic stress did not enhance irp94 mRNA expression, suggesting that irp94 expression was enhanced under ischemic stress and not related to the heat shock signaling mechanism. Our study suggested that irp94, a novel member of the HSP110 family, might play an important role in the environment altering neuronal functions, especially after transient forebrain ischemia.

Animals↗
PubMed1999

Crucial role of group IIA phospholipase A(2) in oleic acid-induced acute lung injury in rabbits.

Group IIA secretory phospholipase A(2) (sPLA(2)) has been implicated in a variety of inflammatory diseases including acute lung injury (ALI); however, the role of sPLA(2) in this disorder remains unclear. The aim of the present investigation was to examine the role of this enzyme in a model of ALI induced by oleic acid (OA) in rabbits by testing human group IIA phospholipase A(2) (PLA(2)) inhibitor, S-5920/LY315920Na. Experimental groups consisted of a saline control group (n = 8), an OA control group (n = 10) infused intravenously with OA (0.1 ml/kg/h for 2 h), and three groups given OA + S-5920/LY315920Na (three different doses, n = 8, respectively). Infusion of OA provoked pulmonary hemorrhage and edema formation, protein leakage, and massive neutrophil infiltration, resulting in severe hypoxemia and impaired lung compliance. PLA(2) activity was detected in the bronchoalveolar lavage fluid (BALF), but not plasma, which correlated well with severity of lung injury in this model. Pretreatment with S-5920/LY315920Na diminished the OA-induced PLA(2) activity in the BALF and dose-dependently attenuated the previously described lung injury induced by OA, accompanied by protection against lung surfactant degradation and production of thromboxane A(2) (TXA(2)) and leukotriene B(4) (LTB(4)). S-5920/LY315920Na also inhibited the OA-induced production of interleukin-8 (IL-8), both in plasma and BALF. Thus, sPLA(2) appears to play a key role in OA-induced lung injury, suggesting that the group IIA PLA(2) inhibitor may be a promising agent for patients with acute respiratory distress syndrome (ARDS).

Acetates↗
PubMed1998

Role of the isoprenyl tail of ubiquinone in reaction with respiratory enzymes: studies with bovine heart mitochondrial complex I and Escherichia coli bo-type ubiquinol oxidase.

The hydrophobic isoprene tail of ubiquinone-2 (Q2) exihibits binding specificity in redox reactions with bovine heart mitochondrial complex I (Ohshima, M., Miyoshi, H., Sakamoto, K., Takegami, K., Iwata, J., Kuwabara, K., Iwamura, H., and Yagi, T. (1998) Biochemistry 37, 6436-6445) and the Escherichia coli bo-type ubiquinol oxidase (Sakamoto, K., Miyoshi, H., Takegami, K., Mogi, T., Anraku, Y., and Iwamura, H. (1996) J. Biol. Chem. 271, 29897-29902). To identify the structural factor(s) of the diprenyl tail of Q2 governing the specific interaction with these enzymes, we synthesized a series of novel Q2 analogues in which only one of the structural factors of the diprenyl tail was systematically modified. In bovine complex I, the presence of the methyl branch and the pi-electron system in the first isoprene unit are responsible for high-affinity binding of Q2 to the ubiquinone reduction site, which results in a low Km and kcat values of Q2 reduction. The position of the methyl group in the tail is strictly recognized by the enzyme. In contrast to complex I, in bo-type ubiquinol oxidase, either of the two pi-electron systems in the tail is required for high-affinity binding of Q2H2 to the enzyme, while the presence of the methyl branch and the location of the pi-electron systems are not strictly recognized by the enzyme. We concluded that the role of the ubiquinone tail is not simply the enhancement of the hydrophobicity of the molecule and that molecular recognition of the tail by the quinone redox site differs among the respiratory enzymes.

Animals↗
PubMed1998

Marked, sustained expression of a novel 150-kDa oxygen-regulated stress protein, in severely ischemic mouse neurons.

The 150-kDa oxygen-regulated protein (ORP150) first was described with reference to the central nervous system in cultured astrocytes subjected to dense hypoxia. Subsequently its transcript was found in macrophages within human aortic atherosclerotic plaques, suggesting a role in protecting cells under hypoxic stress. In a mouse model of permanent focal brain ischemia, we aimed to elucidate the constitutive cellular localization in vivo of ORP150 in the central nervous system as well as the sequential alteration in its mRNA and protein expression during this severe ischemic insult. Immunohistochemical study demonstrated that ORP150 protein normally is present predominantly in neurons. The 78-kDa glucose-regulated protein, which is another well-known stress protein retained in the endoplasmic reticulum, also was stained in neurons. During the first 3 h after ischemia, ORP150 antigenicity was markedly enhanced in severely damaged neurons, while the amount of the glucose-regulated protein was decreased. Preceding this change, orp150 mRNA was selectively induced in neurons undergoing postischemic cytoskeletal proteolysis, as early as 1 h after middle cerebral artery occlusion. These results indicated that ORP150 might be regulated by transcriptional level as for many stress proteins, but unlike previously described other stress proteins it was translated in the center of ischemic lesions despite nearly complete energy depletion. In this paper, the biological potentials of ORP150 protein in the setting of brain ischemia in vivo will also be discussed.

Animals↗
PubMed1998

Characterization of the ubiquinone reduction site of mitochondrial complex I using bulky synthetic ubiquinones.

A wide variety of alkyl derivatives of Q2 (6-geranyl-2, 3-dimethoxy-5-methyl-1,4-benzoquinone) and DB (6-n-decyl-2, 3-dimethoxy-5-methyl-1,4-benzoquinone), in which methoxy groups of the 2- and/or 3-positions of the quinone ring were replaced by other bulky alkoxy groups from ethoxy to butoxy, were prepared by novel synthetic procedures. Electron-accepting activities of the bulky quinones were investigated with bovine heart mitochondrial complex I and its counterpart of Paracoccus denitrificans(NDH-1) to elucidate structural and functional features of the quinone reduction site of the enzymes. The bulky quinone analogues served as sufficient electron acceptors from the physiological quinone reduction site of bovine complex I. Considering the very poor activities of even the ethoxy derivatives as substrates for other respiratory enzymes such as mitochondrial complexes II and III [He, D. Y., Gu, L. Q., Yu, L., and Yu, C. A. (1994) Biochemistry 33, 880-884], this result indicated that the quinone reduction site of bovine complex I is spacious enough to accommodate bulky exogenous substrates. In contrast to bovine complex I, bulky quinone analogues served as poor electron acceptors with Paracoccus NDH-1. These observations indicated that bovine complex I recognizes the substrate structure with poor specificity. The substituent effects in the 2- and 3-positions of the quinone ring on the electron-transfer activity with bovine complex I differed significantly between Q2 and DB series despite having the same total number of carbon atoms in the side chain. The inhibitory effect involving Q2 due to its geranyl side chain was markedly diminished by structural modifications of the quinone ring moiety. These findings indicate that the side chain plays a specific role in the redox reaction and that the quinone ring and side-chain moieties contribute interdependently to binding interaction. Moreover, structural dependency of the proton-pumping activity of the quinone analogues was comparable to that of the electron-transfer activity with bovine complex I, indicating that the mechanism of redox-driven proton-pumping does not differ depending upon the substrate structure.

Animals↗
PubMed1998

Difference in chromatin packaging between active and inactive X chromosomes by fractionation and allele-specific detection.

Using a novel method consisting of chromatin fractionation and allele-specific detection, chromatin packaging is compared between active X (Xa) and inactive X (Xi) chromosomes for five tumor cell clones that were derived from inter-subspecific F1 female mice. Separation of heterochromatic (H) and euchromatic (E) fractions is monitored by hybridization with subtelomeric satellite DNA and ribosomal RNA gene and by PCR amplification of p53 gene/pseudogene with one primer set. The H fraction was enriched with satellite and p53 pseudogene probably existing in heterochromatic regions while the E fraction showed inverse, suggesting fair separation. Analysis with seven marker and three gene loci revealed concentration of alleles on Xi in the H fraction and those on Xa in the E fraction, though the concentration levels varied. This implies that the packaging level of Xi is higher than that of active or inactive euchromatin on Xa. Intriguingly, one cell line showed biallelic expression and chromatin relaxation of the Pgk-1 locus, suggesting that the relaxation occur regionally on X chromosome.

Alleles↗
PubMed1998

Induction of 72-kDa inducible heat shock protein (HSP72) in cultured rat astrocytes after energy depletion.

Protein synthesis is important in the readaptive processes for cultured astrocytes after hypoxia and subsequent reoxygenation. We have identified 72-kDa inducible heat shock protein (HSP72) as a major stress protein in reoxygenated astrocytes. To assess the mechanism for reoxygenation-mediated induction of HSP72, a reporter gene that consists of a human HSP promoter fused to the luciferase gene was transfected into cultured astrocytes. Analysis of cellular energy nucleotides showed an increase of the ADP/ATP ratio after reoxygenation, which synchronized with activation of the HSP promoter. Activation of the HSP promoter was also observed after an addition of iodoacetic acid to hypoxic astrocytes, which reached the maximum when the ADP/ATP ratio reached 50%, but further decline in the energy profile caused inactivation of this promoter. Inhibition of protein synthesis after reoxygenation resulted in temporary restoration of the energy profile and suppression of the DNA binding activity of the heat shock factor. Addition of quercetin greatly decreased the [3H]leucine incorporation in the polysome fraction without any effect on the mature mRNA formation. These data suggest that the energy depletion in reoxygenation triggers induction of HSP72 after reoxygenation, which may act as a pivotal mediator in the stress response of reoxygenated astrocytes by facilitating protein synthesis.

Adenine Nucleotides↗
PubMed1998

Amelioration of hippocampal neuronal damage after global ischemia by neuronal overexpression of BCL-2 in transgenic mice.

BACKGROUND AND PURPOSE: Reports suggesting the involvement of apoptosis in ischemic neuronal damage have been accumulating, and protection against apoptotic death by BCL-2 has been shown in many types of cells. Overexpression of BCL-2 has been shown to reduce infarct size after focal ischemia. The purpose of the present study was to assess whether BCL-2 exerted its effect on selective neuronal vulnerability after transient global ischemia. METHODS: Transgenic mice overexpressing BCL-2 in neurons and their littermates were subjected to transient forebrain ischemia for 12 minutes, and the hippocampus was examined 7 days later with conventional histology, immunohistochemistry, and in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling of fragmented DNA. RESULTS: Although both types of mice showed a similar degree of ischemic insult, transgenic mice showed a lesser degree of neuronal death together with DNA fragmentation in the hippocampus than their littermates. CONCLUSIONS: Overexpression of BCL-2 in neurons mitigates selective neuronal vulnerability in the hippocampus of transgenic mice after transient global ischemia.

Animals↗
PubMed1998

[DNA bend sites in the promoter region of the human estrogen receptor alpha gene].

DNA bend sites in the promoter region of the human estrogen receptor a gene were determined by the circular permutation assay. Among a total of five sites (ERB -4 to -1, and ERB + 1) mapped in the 3 kb region, three matched with the positions of the predicted periodicity while the other two did not. Most of the sites were accompanied by the short poly (dA)-poly (dT) tracts including the potential bend core sequence A2N8A2N8A2 (A/A/A). Fine mapping of the ERB-2 site indicated that this A/A/A and the immediate franking sequences contained motifs for the estrogen response element. This region had a higher affinity for the nuclear scaffold and was included in the core region of the nucleosome structure. However, binding of the nuclear factor(s) to the motifs and disruption of nucleosome structure occurred without ATP. These results suggest that a class of periodic bent DNA could act as a site of multiple interactions among the nuclear scaffold, core histones and nuclear factors.

DNA↗