Ryoji Konishi

Localization and developmental changes in the neuron-specific cyclin-dependent kinase 5 activator (p35nck5a) in the rat brain

Mammalian brains contain a cde2-like protein kinase which is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a brain-specific regulatory subunit with a molecular weight of 35,000. In this study, we examined the temporal and spatial expression patterns of p35nck5a in the developing rat brain. Northern blot analysis showed that p35nck5a messenger RNA expression was low in the brain of 12-day postcoitum rats, and increased to a much higher level from 18 days postcoitum to two weeks after birth, and then declined at three weeks after birth. These developmental changes in p35nck5a expression correlated with the changes in Cdk5-associated kinase activity during brain development. These data suggest that p35nck5a is the specific activator for Cdk5 in the brain. Immunohistochemical and in situ hybridization studies demonstrated the presence of p35nck5a protein in postmitotic neurons but not in glial cells at all stages of brain development, indicating that p35nck5a is a neuron-specific protein. In the adult brain, the protein was rich in cell bodies and dendrites, and only very low amounts were detected in axons. In fetal and neonatal brains, however, axonal pathways such as the corpus callosum and external capsule were also stained with anti-p35nck5a antibody. Our findings suggest that p35nck5a is neuron specific, and a specific activator for Cdk5, and the subcellular localization of the two is strictly regulated depending on brain development. Neuronal Cdc2-like kinase may play key roles in neuronal maturation, synaptic formation, and neuronal plasticity.

Suppression of hyperemia and DNA oxidation by indomethacin in cerebral ischemia

We investigated antioxidative activity and the effect of indomethacin, an agent that inhibits cyclooxygenase, on extracellular glutamate and cerebral blood flow in cerebral ischemia in gerbils. Pre-ischemic administration of indomethacin (5 mg/kg, i.p.) significantly rescued hippocampal CA1 neurons (9+/-6 cells/mm in the ischemia, 87+/-43 cells/mm in the indomethacin group, P<0.001). DNA fragmentation induced by ischemia was also examined using the terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) method and indomethacin reduced TUNEL positive cells (140+/-21 in the ischemia, 99+/-31 in the indomethacin group, P<0.01). In addition, indomethacin attenuated the increase in hippocampal blood flow during reperfusion, but not increased extracellular glutamate by ischemia. Eight-hydroxydeoxyguanosine (8-OH-dG), a highly sensitive marker of DNA oxidation, was measured 90 min following ischemia using high-pressure liquid chromatography. Indomethacin significantly decreased the level of ischemia-induced 8-OH-dG in the hippocampus (P<0.05). These results suggest that indomethacin may protect neurons by attenuating oxidative stress and reperfusion injury in ischemic insult.

Involvement of calmodulin-dependent protein kinases-I and -IV in long-term potentiation.

Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) are thought to be involved in the induction of long-term potentiation (LTP). In the present study, LTP was induced by theta burst stimulation in the Schaffer collateral area of the stratum radiatum in the hippocampal CA1 region of the rat hippocampus. LTP-induced and control hippocampal slices were studied by Western blot and immunohistochemical analyses using CaMK-I, -II and -IV antibodies. Increased amounts of all three CaMKs were found in LTP-induced hippocampal slices as indicated by Western blot as well as by the density of their immunoreactivity. Our data clearly shows that not only CaMK-II but also CaMK-I and -IV contribute to synaptic plasticity formed in LTP.

Morphological changes in the hippocampus in amygdaloid kindled mouse

To clarify the origin and maintenance of epileptogenesis, morphological changes in the hippocampus of amygdaloid-kindled mice were analyzed at different stages of kindling. The granule cell size in dentate gyrus and the pyramidal cell size in CA1 were clearly decreased depending on seizure stage. The cell size in CA2 was increased and density in dentate gyrus and CA2 was reduced, significantly. The morphological changes in hippocampus associated with kindling must be closely related to the acquisition and the maintenance of epileptogenesis. The results support the hypothesis that seizure-induced damage of neurons may lead to formation of new synaptic connections that produce abnormal hyperexitability and result in seizures.

Analysis of the inhibitory mechanism of D-allose on MOLT-4F leukemia cell proliferation

D-Allose, the C-3 epimer of D-glucose, is one of the rare sugars found in nature. In the present study, we have elucidated for the first time that various leukemia cell lines have different susceptibility to anti-proliferative activity of D-allose, and that this difference is related to the difference in induction of thioredoxin interacting protein (TXNIP) expression. We examined 5 leukemia cell lines (MOLT-4F, IM-9, HL-60, BALL-1 and Daudi), and found that MOLT-4F (T-cell lymphoblastic leukemia) had the highest susceptibility to D-allose, and that Daudi (Burkitts lymphoma) had the lowest. D-Allose significantly slowed the cell cycle progression without causing apoptosis of MOLT-4F cells. Intracellular TXNIP expression was specifically and markedly enhanced in MOLT-4F cells by D-allose treatment, and subsequent increase of p27(kip1), a cell cycle inhibitor, was observed. On the other hand, D-allose did not increase TXNIP and p27(kip1) levels at all in Daudi cells. These results indicate that D-allose suppresses MOLT-4F cell proliferation possibly by the inhibition of cell cycle progression via induction of TXNIP expression.

Overexpression of cyclin E and cyclin-dependent kinase 2 is correlated with development of hepatocellular carcinomas

Mammalian cell cycle progression is regulated by the combined action of cyclins/cyclin-dependent kinases (CDKs) and CDK inhibitors. Abnormal expression as well as interaction of these proteins may result in malignant transformation of cells. To further address the role of these cell cycle proteins in hepatocellular carcinomas, we analyzed the expression of cyclin E and CDK2. A panel of livers with human hepatocellular carcinoma, liver cirrhosis, and chronic hepatitis were used as a human experimental system. The inbred LEC (Long-Evans with a cinnamon-like coat color) rats were used as an animal experimental HCC model. Immunohistochemical staining of serial paraffin sections was performed using antibodies to cyclin E and CDK2. The results showed that cyclin E and CDK2 were concurrently overexpressed in hepatocellular carcinomas both in human and rat livers. Western blot analysis and CDK2 kinase assay demonstrated expression levels of cyclin E and CDK2 and CDK2 kinase activity, respectively, and both were shown to increase along with the development of hepatocellular carcinomas. Analysis of the correlation between expression of cyclin E and CDK2 and clinicopathological parameters revealed a significant correlation between expression of cyclin E and tumor grade (P=0.013), and PCNA index (P=0.006) as well as CDK2 expression (P=0.015). Overexpression of CDK2 tended to be associated with poorly differentiated HCCs. The results suggest that overexpression of cyclin E and CDK2 plays an important role in the development of hepatocellular carcinoma.

Calbrain, a Novel Two EF-hand Calcium-binding Protein That Suppresses Ca2+/Calmodulin-dependent Protein Kinase II Activity in the Brain

A cDNA clone that encodes a novel Ca2+-binding protein was isolated from a human brain cDNA library. The gene for this clone, termed calbrain, encodes a 70-amino acid polypeptide with a predicted molecular mass of 8.06 kDa. The analysis of deduced amino acid sequence revealed that calbrain contains two putative EF-hand motifs that show significantly high homology to those of the calmodulin (CaM) family rather than two EF-hand protein families. By Northern hybridization analysis, an approximate 1.5-kilobase pair transcript of calbrain was detected exclusively in the brain, and in situ hybridization study revealed its abundant expression in the hippocampus, habenular area in the epithalamus, and in the cerebellum. A recombinant calbrain protein showed a Ca2+ binding capacity, suggesting the functional potency as a regulator of Ca2+-mediated cellular processes. Ca2+/calmodulin-dependent kinase II, the most abundant protein kinase in the hippocampus and strongly implicated in the basic neuronal functions, was used to evaluate the physiological roles of calbrain. Studies in vitro revealed that calbrain competitively inhibited CaM binding to Ca2+/calmodulin-dependent kinase II (K i = 129 nm) and reduced its kinase activity and autophosphorylation.

Stage- and region-specific cyclooxygenase expression and effects of a selective COX-1 inhibitor in the mouse amygdala kindling model

In an attempt to elucidate the involvement of cyclooxygenase (COX) enzymes, particularly COX-1, in epileptogenesis, the localization of COX-1 and COX-2 expression in the mouse kindling model was analyzed by immunohistochemistry. COX-2 was predominantly observed in brain neurons and its concentration in the hippocampus increased with progressing seizures, as reported previously. COX-1 was predominant in microglia and its concentration was also enhanced in the hippocampus and areas around the third ventricle during the progression of seizures. These regions are thought to play an important role in the propagation of limbic seizures. Moreover, the administration of SC-560 (a selective COX-1 inhibitor) or indomethacin (a non-selective COX inhibitor) retarded the progress of seizures. Although the precise function of COX-positive cells in microglia and elsewhere is not clear, our results suggest that COX-1 as well as COX-2 may be involved in epileptogenesis, and that certain COX inhibitors can potentially prevent the occurrence of seizures.

Expression and subcellular localization of multifunctional calmodulin-dependent protein kinases-I, -II and -IV are altered in rat hippocampal CA1 neurons after induction of long-term potentiation.

Long-term potentiation (LTP) is considered to be associated with an increase in expression as well as activity of Ca(2+)/calmodulin-dependent protein kinases (CaMKs). LTP-induced and control hippocampal slices were studied by immunohistochemical and electronmicroscopic analyses using anti-CaMK-I, -II and -IV antibodies. All three kinases were demonstrated to increase their expression in CA1 neurons. CaMK-I was shown to mainly localize in the cytoplasm of the control and LTP-induced neurons, and a significant increase of immunoreactivity was observed in the latter neurons. A part of CaMK-I was found to translocate to the nuclei of LTP-induced hippocampal CA1 neurons. Direct evidence of the translocation of CaMK-II from cytoplasm to nuclei in LTP was demonstrated by immuno-electronmicroscopy. A significant increase in expression of CaMK-IV in the nuclei was also observed. Our data suggest that all the three CaMKs were actively involved in nuclear Ca(2+)-signaling in LTP.

A novel nucleic acid analogue shows strong angiogenic activity

A novel nucleic acid analogue (2Cl-C.OXT-A) significantly stimulated tube formation of human umbilical endothelial cells (HUVEC). Its maximum potency at 100muM was stronger than that of vascular endothelial growth factor (VEGF), a positive control. At this concentration, 2Cl-C.OXT-A moderately stimulated proliferation as well as migration of HUVEC. To gain mechanistic insights how 2Cl-C.OXT-A promotes angiogenic responses in HUVEC, we performed immunoblot analyses using phospho-specific antibodies as probes. 2Cl-C.OXT-A induced robust phosphorylation/activation of MAP kinase ERK1/2 and an upstream MAP kinase kinase MEK. Conversely, a MEK inhibitor PD98059 abolished ERK1/2 activation and tube formation both enhanced by 2Cl-C.OXT-A. In contrast, MAP kinase responses elicited by 2Cl-C.OXT-A were not inhibited by SU5416, a specific inhibitor of VEGF receptor tyrosine kinase. Collectively these results suggest that 2Cl-C.OXT-A-induces angiogenic responses in HUVEC mediated by a MAP kinase cascade comprising MEK and ERK1/2, but independently of VEGF receptor tyrosine kinase. In vivo assay using chicken chorioallantoic membrane (CAM) and rabbit cornea also suggested the angiogenic potency of 2Cl-C.OXT-A.