Biomedical subjects

K Yamada

Publications and source records attributed to K Yamada.

At least 361 records · Page 20Linked to original sources

PubMed2000

Co-induction of heme oxygenase-1 and peroxiredoxin I in astrocytes and microglia around hemorrhagic region in the rat brain.

Heme[none1] oxygenase-1 (HO-1) and peroxiredoxin I (PrxI) are known to be oxidative stress- and heme-related proteins. The antioxidant activity of PrxI is inhibited by heme, therefore co-expression of HO-1 and PrxI is considered to be a reasonable mechanism to maintain its antioxidative function. Immunoblotting demonstrated that HO-1 and PrxI were induced around the hemorrhagic region. Immunohistochemical studies revealed that, in acute phase, HO-1 and PrxI were induced primarily in microglia. In the subacute and chronic phase, the immunoreactivity of HO-1 and PrxI in astrocytes was the most intense. These data are the first to demonstrate co-induction of HO-1 and PrxI in the brain. Our results suggest that HO-1 and PrxI are localized in a similar manner to assure the antioxidant activity of PrxI under stress conditions associated with intracerebral hemorrhage.

Animals↗

PubMed2000

Unique oxidative mechanisms for the reactive nitrogen oxide species, nitroxyl anion.

The nitroxyl anion (NO-) is a highly reactive molecule that may be involved in pathophysiological actions associated with increased formation of reactive nitrogen oxide species. Angeli's salt (Na2N2O3; AS) is a NO- donor that has been shown to exert marked cytotoxicity. However, its decomposition intermediates have not been well characterized. In this study, the chemical reactivity of AS was examined and compared with that of peroxynitrite (ONOO-) and NO/N2O3. Under aerobic conditions, AS and ONOO- exhibited similar and considerably higher affinities for dihydrorhodamine (DHR) than NO/N2O3. Quenching of DHR oxidation by azide and nitrosation of diaminonaphthalene were exclusively observed with NO/N2O3. Additional comparison of ONOO- and AS chemistry demonstrated that ONOO- was a far more potent one-electron oxidant and nitrating agent of hydroxyphenylacetic acid than was AS. However, AS was more effective at hydroxylating benzoic acid than was ONOO-. Taken together, these data indicate that neither NO/N2O3 nor ONOO- is an intermediate of AS decomposition. Evaluation of the stoichiometry of AS decomposition and O2 consumption revealed a 1:1 molar ratio. Indeed, oxidation of DHR mediated by AS proved to be oxygen-dependent. Analysis of the end products of AS decomposition demonstrated formation of NO2- and NO3- in approximately stoichiometric ratios. Several mechanisms are proposed for O2 adduct formation followed by decomposition to NO3- or by oxidation of an HN2O3- molecule to form NO2-. Given that the cytotoxicity of AS is far greater than that of either NO/N2O3 or NO + O2, this study provides important new insights into the implications of the potential endogenous formation of NO- under inflammatory conditions in vivo.

Nitrates↗

PubMed2000

L-serine and glycine serve as major astroglia-derived trophic factors for cerebellar Purkinje neurons.

Glial cells support the survival and development of central neurons through the supply of trophic factors. Here we demonstrate that l-serine (l-Ser) and glycine (Gly) also are glia-derived trophic factors. These amino acids are released by astroglial cells and promote the survival, dendritogenesis, and electrophysiological development of cultured cerebellar Purkinje neurons. Although l-Ser and Gly are generally classified as nonessential amino acids, 3-phosphoglycerate dehydrogenase (3PGDH), a key enzyme for their biosynthesis, is not expressed in Purkinje neurons. By contrast, the Bergman glia, a native astroglia in the cerebellar cortex, highly expresses 3PGDH. These data suggest that l-Ser and Gly mediate the trophic actions of glial cells on Purkinje neurons.

Animals↗

PubMed2000

Homeobox gene Hex is essential for onset of mouse embryonic liver development and differentiation of the monocyte lineage.

Disruption of the mouse Hex gene resulted in embryonic lethality around embryonic age (E) 10.5, due to no substantial liver formation. Expression of albumin was detectable in heterozygous (Hex(+/-)) but not in homozygous (Hex(-/-)) [corrected] embryos at E8.5. Instead of liver bud formation at E9.5, a liver-like capsule structure was observed in Hex(-/-) [corrected] embryos. In Hex(-/-) [corrected] mutant liver, we found no hepatocytes but no signs of apoptotic cell death in the area. Expression of transcription factors involved in hepatocyte differentiation, hepatocyte nuclear factor (Hnf)3beta, Hnf6, Hnf4alpha and Hnf1alpha, were restricted to the capsule and internal matrix-like structure in the mutant liver and expression of a subset of these factors were reduced. Hematopoiesis of monocytes was impaired in mutant embryos while erythroid lineage was unaffected. These results indicate that Hex plays an essential role in progenitor cells which commit to the hepatic endoderm and in the hematopoietic differentiation of the monocyte lineage.

Albumins↗

PubMed2000

Accumulation of phosphorylated neurofilaments and increase in apoptosis-specific protein and phosphorylated c-Jun induced by proteasome inhibitors.

The ubiquitin-proteasome system has been regarded as being important in the progression of neurodegenerative diseases, although its exact role remains uncertain. This in vitro study using PC12h cell cultures examined whether interference with the ubiquitin-proteasome system by proteasome inhibitors induces the neuropathological features of neurodegenerative diseases. Perikaryal accumulation of phosphorylated neurofilaments and an increase in c-Jun as well as phosphorylated form of c-Jun and apoptosis-specific protein were induced by the proteasome inhibitors lactacystin and N-carbobenzoxy-leucyl-leucyl-leucinal. These changes were not observed when only calpain was inhibited. The present study therefore suggests the possibility that a perturbation of the ubiquitin-proteasome system may be one of the causes that result in the development of neuropathological features. Additionally, activity assays showed that the proteasome inhibitor caused an increase in the activity of c-Jun N-terminal kinase (JNK/SAPK), which can phosphorylate neurofilaments and c-Jun, suggesting the possible involvement of JNK in phosphorylation of these proteins.

Acetylcysteine↗

PubMed2000

Reduction of myocardial infarct size by SM-20550, a novel Na(+)/H(+) exchange inhibitor, in rabbits.

The effects of N-(aminoiminomethyl)-1, 4-dimethyl-1H-indole-2-carboxamide methanesulfonic acid (SM-20550), a novel potent Na(+)/H(+) exchanger, and nicorandil, a K(+) channel opener with nitrate-like activity, were studied in a myocardial ischemia and reperfusion injury model. Anesthetized rabbits underwent occlusion of the coronary artery (30 min) followed by reperfusion (5 h). Intravenous administration of SM-20550 before ischemia reduced the infarct size by approximately 30-70% in a dose-dependent manner, with a significant reduction in serum creatine phosphokinase activity. Similarly, intravenous administration of nicorandil before ischemia reduced the infarct size by 33% with a significant reduction in serum creatine phosphokinase activity. Moreover, intravenous administration of SM-20550 after ischemia resulted in a significant, approximately 20-40% reduction in the infarct size, but the administration of nicorandil after ischemia did not reduce the infarct size. These results indicate that SM-20550 reduced myocardial necrosis when administered either before or after ischemia.

Amidines↗

PubMed2000

Spatiotemporal expression of BDNF in the hippocampus induced by the continuous intracerebroventricular infusion of beta-amyloid in rats.

The beta-amyloid protein (Abeta) is the major component of senile plaques found in the brain in Alzheimer's disease (AD). Its neurotoxic properties in vivo, however, are not well defined. Since the expression of neurotrophin genes is considered an important component of the intrinsic neuroprotective response to insults, we analyzed the gene expression of neurotrophins in the brains of rats which received a continuous infusion of Abeta-(1-42) into the cerebroventricle. Northern blot analysis revealed a significant increase in brain-derived neurotrophic factor (BDNF) expression in the hippocampus but no change in the cerebral cortices. The alteration peaked on days 3-7 and returned to the basal level on day 14 after the start of Abeta-(1-42) infusion. No significant changes in nerve growth factor or neurotrophin-3 mRNA expression were observed. The infusion of Abeta-(1-40) and (25-35) also triggered the expression of BDNF mRNA, whereas neither Abeta-(40-1) nor (1-16) had any effect. In situ hybridization histochemistry revealed that the expression mainly occurred in the hilus and granular layer of the dentate gyrus and to a lesser extent in the pyramidal neurons of the CA1 region. These results demonstrate that the continuous intracerebroventricular infusion of Abeta induces selective and spatiotemporal expression of BDNF mRNA in the hippocampus.

Alzheimer Disease↗

PubMed2000

Involvement of brain-derived neurotrophic factor in spatial memory formation and maintenance in a radial arm maze test in rats.

Brain-derived neurotrophic factor (BDNF) regulates both short-term synaptic functions and activity-dependent synaptic plasticity such as long-term potentiation. In the present study, we investigated the role of BDNF in the spatial reference and working memory in a radial arm maze test. The radial arm maze training resulted in a significant increase in the BDNF mRNA expression in the hippocampus, although the expression in the frontal cortex did not change. When spatial learning was inhibited by treatment with 7-nitroindazole, an inhibitor of brain nitric oxide synthase, the increase in the hippocampal BDNF mRNA did not occur. To clarify the causal relation between BDNF mRNA expression and spatial memory formation, we examined the effects of antisense BDNF treatment on spatial learning and memory. A continuous intracerebroventricular infusion of antisense BDNF oligonucleotide resulted in an impairment of spatial learning, although the sense oligonucleotide had no effect. Treatment with antisense, but not sense, BDNF oligonucleotide was associated with a significant reduction of BDNF mRNA and protein levels in the hippocampus. Furthermore, treatment with antisense BDNF oligonucleotide in rats, which had previously acquired spatial memory by an extensive training, impaired both reference and working memory. There were no differences in locomotor activity, food consumption, and body weight between the antisense and sense oligonucleotide-treated rats. These results suggest that BDNF plays an important role not only in the formation, but also in the retention and/or recall, of spatial memory.

Analysis of Variance↗

PubMed2000

Characterization of the human NTAK gene structure and distribution of the isoforms for rat NTAK mRNA.

NTAK (neural- and thymus-derived activator for the ErbB kinase, neuregulin-2) is a novel member of the epidermal growth factor (EGF) family. We have isolated and characterized the human NTAK gene, comprising 12 exons spanning in excess of 55 kilobases (kb). The 7. 0kb long mRNA of the human NTAK gene was expressed in the human neuroblastoma SK-N-SH cell line with two alternative isoforms detected. Furthermore, six isoforms have been identified from rat brain and PC-12 cells. Although the alpha isoform of the NTAK gene was found to be expressed in all tissues including brain, the beta isoform was expressed only in rat brain tissues. Potential regulatory regions included consensus binding sites for AP-2, TF-IIIA, Sp-1, and YY-1 located in the 5'-flanking region of the NTAK gene.

Amino Acid Sequence↗

PubMed2000

Characterization of mSelB, a novel mammalian elongation factor for selenoprotein translation.

Decoding of UGA selenocysteine codons in eubacteria is mediated by the specialized elongation factor SelB, which conveys the charged tRNA(Sec) to the A site of the ribosome, through binding to the SECIS mRNA hairpin. In an attempt to isolate the eukaryotic homolog of SelB, a database search in this work identified a mouse expressed sequence tag containing the complete cDNA encoding a novel protein of 583 amino acids, which we called mSelB. Several lines of evidence enabled us to establish that mSelB is the bona fide mammalian elongation factor for selenoprotein translation: it binds GTP, recognizes the Sec-tRNA(Sec) in vitro and in vivo, and is required for efficient selenoprotein translation in vivo. In contrast to the eubacterial SelB, the recombinant mSelB alone is unable to bind specifically the eukaryotic SECIS RNA hairpin. However, complementation with HeLa cell extracts led to the formation of a SECIS-dependent complex containing mSelB and at least another factor. Therefore, the role carried out by a single elongation factor in eubacterial selenoprotein translation is devoted to two or more specialized proteins in eukaryotes.

Amino Acid Sequence↗

PubMed2000

Constitutive activation of STAT5 by a point mutation in the SH2 domain.

We previously identified a constitutively active form of STAT (signal transducer and activator of transcription) 5A by polymerase chain reaction-driven random mutagenesis followed by retrovirus-mediated expression screening, which had two point mutations in the DNA-binding and transcriptional activation domains, and was designated STAT5A1*6. STAT5A1*6 showed markedly elevated DNA binding and transactivation activities with stable tyrosine phosphorylation and nuclear accumulation, and conferred autonomous cell growth on interleukin 3-dependent Ba/F3 cells. We now report another constitutively active mutant, STAT5A-N642H which has a single point mutation (N642H) in its SH2 domain, identified using the same strategy as that used to identify STAT5A1*6. STAT5A-N642H showed identical properties to those of STAT5A1*6 both biochemically and biologically. Interestingly the mutation in STAT5A-N642H resulted in restoration of the conserved critical histidine which is involved in the binding of phosphotyrosine in the majority of SH2-containing proteins. Introduction of an additional mutation (Y694F) to STAT5A-N642H, which disrupted critical tyrosine 694 required for dimerization of STAT5, abolished all the activities manifested by the mutant STAT5A-N642H, which indicates that dimerization is required for the activity of STAT5A-N642H as was the case for the wild-type STAT5A. The present findings also show that different mutations rendered STAT5A constitutively active, through a common mechanism, which is similar to that of physiological activation.

3T3 Cells↗

PubMed2000

Transcriptional regulation of rat scavenger receptor class B type I gene.

The scavenger receptor class B type I (SR-BI) mediates the selective transport of lipids from high density lipoprotein to cells and plays an important role in the reverse uptake of cholesterol to the liver and in the delivery of substrates for steroidogenesis in steroidogenic organs. We report here on the isolation and characterization of the upstream promoter region of the rat SR-BI gene. The transcription start site for rat SR-BI was mapped, and DNA sequence analysis revealed the presence of binding sites for the Sp1 family in the proximal 5'-flanking region. Analysis of deletion mutants with different 5' lengths revealed that the region between -121 and -90 base pairs from the transcription start site is essential for the efficient transcription of SR-BI. Both Sp1 and Sp3 bind to three GC boxes in the region (-141 to -1 base pairs) in a sequence-specific manner. Mutations in any of the GC boxes decreased efficient transcription from this promoter in MA-10 mouse Leydig tumor cells. The overexpression of Sp1 or Sp3 protein enhanced the rat SR-BI promoter activity. These results indicate that Sp1 family members of transcription factors are essential for transcription of the rat SR-BI gene.

Animals↗

PubMed2000

Measurement of glucose uptake and intracellular calcium concentration in single, living pancreatic beta-cells.

There has been no method previously to measure both glucose transport and its effect on the various intracellular functions in single, living mammalian cells. A fluorescent derivative of d-glucose, 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), that we have developed has made such measurements possible. COS-1 cells that overexpress the human glucose transporter GLUT2 show significantly greater 2-NBDG uptake than mock transfected cells. Using GLUT2-abundant mouse insulin-secreting clonal MIN6 cells, we found that 2-NBDG was incorporated into the cells in a time- and concentration-dependent manner. The 2-NBDG uptake was inhibited by high concentrations of d-glucose in a dose-dependent manner and also was almost completely inhibited by 10 micrometer cytochalasin B. We then measured both glucose uptake and the intracellular calcium concentration ([Ca(2+)](i)) in single, living pancreatic islet cells. 2-NBDG and fura-2 were used as the tracer of glucose and indicator of intracellular calcium, respectively. All of the cells that showed an increase in [Ca(2+)](i) in response to a high concentration of glucose (16.8 mm) rapidly incorporated significant 2-NBDG. Immunocytochemical examination confirmed these cells to be insulin-positive beta-cells. All of the cells that showed no significant, rapid 2-NBDG uptake lacked such glucose responsiveness of [Ca(2+)](i), indicating that these cells were non-beta-cells such as glucagon-positive alpha-cells. These results show the uptake of glucose causing a concomitant increase of [Ca(2+)](i) in beta-cells. Because 2-NBDG is incorporated into mammalian cells through glucose transporters, it should be useful for the measurement of glucose uptake together with concomitant intracellular activities in many types of single, living mammalian cells.

Animals↗

PubMed2000

p53-independent transient p21(WAF1/CIP1) mRNA induction in the rat brain following experimental traumatic injury.

The expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) mRNA after traumatic brain injury in rats was investigated using an in situ hybridization technique, along with regulating gene p53 and stress response gene hsp70 mRNA levels. At 3 h postinjury, p21(WAF1/CIP1) mRNA was markedly increased in the cortex, white matter, thalamus, CA2, a part of CA1,3 and dentate gyrus of the injured side. Hybridization signals remained elevated at 6 h in injured cortex and hippocampus and returned to the baseline by 24 h post-insult. On the other hand, p53 mRNA induction was not observed in any brain sections throughout the post-injury time course. Slight expression of hsp70 mRNA was detected in the injured cortex 3-6 h following injury and this was similar to the temporary pattern of p21(WAF1/CIP1) mRNA expression. This study showed p21(WAF1/CIP1) mRNA to be transiently induced after traumatic brain injury, independent of p53, this possibly being an early stress response to protect cells by arresting them in the cycle and allow DNA repair.

Animals↗

PubMed2000

Isolation and characterization of three novel serine protease genes from Xenopus laevis.

Three novel cDNAs encoding serine proteases, that may play a role in early vertebrate development, have been identified from Xenopus laevis. These Xenopus cDNAs encode trypsin-like serine proteases and are designated Xenopus embryonic serine protease (Xesp)-1, Xesp-2, and XMT-SP1, a homolog of human MT-SP1. Xesp-1 is likely to be a secreted protein that functions in the extracellular space. Xesp-2 and XMP-SP1 are likely to be type II membrane proteases with multidomain structures. Xesp-2 has eight low density lipoprotein receptor (LDLR) domains and one scavenger receptor cysteine-rich (SRCR) domain, and XMT-SP1 has four LDLR domains and two CUB domains. The temporal expressions of these serine protease genes show distinct and characteristic patterns during embryogenesis, and they are differently distributed in adult tissues. Overexpression of Xesp-1 caused no significant defect in embryonic development, but overexpression of Xesp-2 or XMT-SP1 caused defective gastrulation or apoptosis, respectively. These results suggest that these proteases may play important roles during early Xenopus development, such as regulation of cell movement in gastrulae.

Amino Acid Sequence↗

PubMed2000

Effects of sigma(1) receptor agonist SA4503 and neuroactive steroids on performance in a radial arm maze task in rats.

This study examined the effects of sigma(1) receptor agonist SA4503 and neuroactive steroids dehydroepiandrosterone sulfate (DHEAS), pregnenolone sulfate (PREGS) and progesterone (PROG) on spatial working and reference memory in a radial arm maze task in rats. The insertion of a 6-min delay between the 2nd and 3rd choices caused a specific decline in working memory, but had no effect on reference memory. This decline in working memory was improved by SA4503, but not by DHEAS, PREGS or PROG. A non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine significantly impaired both working and reference memory in the presence or absence of a delay. The dizocilpine-induced impairments in the presence of a 6-min delay were ameliorated by SA4503, DHEAS and PREGS, whereas PROG had no effect. The beneficial effects of SA4503, DHEAS and PREGS were antagonized by treatment with sigma(1) receptor antagonist N, N-dipropyl-2-(4-methoxy-3-(2-phenylethoxy)phenyl)-ethylamine hydrochloride (NE-100). Furthermore, PROG attenuated the ameliorating effects of SA4503, DHEAS and PREGS on dizocilpine-induced memory deficits. These results suggest that sigma(1) receptors play a significant role in short-term working memory. Furthermore, it is suggested that DHEAS and PREGS ameliorate dizocilpine-induced memory impairments by acting as sigma(1) receptor agonists, while PROG antagonizes their effects by acting as a sigma(1) receptor antagonist.

Animals↗

PubMed2000

Male mice lacking the gastrin-releasing peptide receptor (GRP-R) display elevated preference for conspecific odors and increased social investigatory behaviors.

Previously, we generated gastrin-releasing peptide receptor null mutant mice (GRP-R-deficient mice), and found that these animals displayed increased non-aggressive social responses in an ordinary social interaction test using a resident-intruder method. In the present study, we examined in more detail the social behaviors of GRP-R-deficient male mice. In social interaction tests, GRP-R-deficient mice showed more social responses, such as sniffing and nosing, relative to wild-type mice, and similar results were obtained whether GRP-R-deficient mice served as intruders or residents. In the same way, they showed more contact behaviors toward an anesthetized conspecific, and less locomotor activity than wild-type mice in a social investigation test toward an anesthetized male mouse. Since olfactory systems play important roles in the social behavior of rodents, olfactory preference tests were conducted in order to evaluate the olfactory properties of GRP-R-deficient mice. The results suggest that no differences exist between wild-type mice and GRP-R-deficient mice in the preference between a novel sawdust odor and their own odor, or that of other male mice. However, GRP-R-deficient mice preferred the odor of other male mice to their own, in contrast to wild-type mice. Furthermore, the preferences of GRP-R-deficient and wild-type mice were not disrupted by intraperitoneal infusion of diazepam (1.5 mg/kg). These results indicate that neither the motion, nor the behavior of conspecifics, nor reduced anxiety lead to the increased non-aggressive social responses and/or social investigatory behaviors in GRP-R-deficient mice. Rather, these latter behaviors may be a consequence of altered cognition of conspecific odors in the mutant mice.

Aggression↗

PubMed2000

Possible association of BLM in decreasing DNA double strand breaks during DNA replication.

Bloom's syndrome (BS) is a rare genetic disorder and the cells from BS patients show genomic instability and an increased level of sister chromatid exchange (SCE). We generated BLM(-/-) and BLM(-/-)/RAD54(-/-) DT40 cells from the chicken B-lymphocyte line DT40. The BLM(-/-) DT40 cells showed higher sensitivity to methyl methanesulfonate and elevated levels of SCE as expected. The targeted integration frequency was also increased remarkably in BLM(-/-) cells. The SCE frequency increase in BLM(-/-) cells was considerably reduced and the enhanced targeted integration observed in BLM(-/-) cells was almost completely abolished in BLM(-/-)/RAD54(-/-) cells, indicating that a large portion of the SCE in BLM(-/-) cells occurs via homologous recombination, and homologous recombination events increase with the defect of BLM function. The BLM(-/-)/RAD54(-/-) cells showed a slow growth phenotype and an increased incidence of chromosome-type breaks/gaps while each single mutant showed relatively small numbers of chromosome-type breaks/gaps.

Adenosine Triphosphatases↗