Junko Sakiyama

Kainic acid-induced convulsions cause prolonged changes in the chondroitin sulfate proteoglycans neurocan and phosphacan in the limbic structures

Systemic administration of kainic acid induces repeated convulsive seizures (KA convulsions) that result in neuropathological changes similar to temporal lobe epilepsy and the appearance of spontaneous recurrent seizures (SRS). The appearance of SRS is considered a result of the remodeling of neuronal networks following neuronal degeneration. We investigated the changes in chondroitin sulfate proteoglycans (CSPGs) in the limbic structures after KA convulsions in the rat using monoclonal antibodies 1G2, which recognizes full-length neurocan and the C-terminal half of neurocan, neurocan C, and 6B4, which recognize phosphacan and protein tyrosine phosphatase zeta. After KA convulsions, full-length neurocan appeared by 24 h and reached a peak by 48 to 72 h, whereas phosphacan decreased within 24 h in the hippocampus. In immunohistochemistry, neurocan increased in the limbic structures coincident with the appearance of reactive astrocytes. Phosphacan decreased coincident with pyramidal cell loss in the hippocampus, and the number of phosphacan-positive perineuronal nets around parvalbumin neurons decreased, whereas parvalbumin neurons were relatively conserved. In contrast, phosphacan increased in the entorhinal and piriform cortices in correlation with the severity of neuronal loss. Both neurocan and phosphacan recovered to the control level by 8 weeks after KA convulsions in some rats, but the changes in neurocan and phosphacan described above still persisted in more than half the rats. The results indicate that KA convulsions induce prolonged changes in neurocan and phosphacan similar to those in the developing rat brain and suggest a role of these CSPGs in the remodeling of neuronal networks related to the establishment or enhancement of epileptogenesis.

Expression of brain specific chondroitin sulfate proteoglycans, neurocan and phosphacan, in the developing and adult hippocampus of Ihara's epileptic rats.

Iharas epileptic rats (IER) is an animal model of temporal lobe epilepsy with mycrodysgenesis, that exhibit abnormal migration of hippocampal neurons and recurrent spontaneous seizures. As an attempt to elucidate the roles of extracellular matrix molecules in the epileptogenecity and mossy fiber sprouting, immunohistochemical localization of brain specific chondroitin sulfate proteoglycans (CSPGs), neurocan and phosphacan, was examined in the hippocampus of postnatal IER and Sprague-Dawley (SD) rats using monoclonal antibodies 1G2 against neurocan and 6B4 against phosphacan. There was no difference in the expression of these two CSPGs between IER and SD rats in the 1st postnatal week. However, the expression of neurocan was poor in the hippocampus of IER in the 2nd and 3rd weeks whereas intense labeling of neurocan was present throughout the hippocampus of SD rats. Labeling of neurocan was almost absent in the hippocampus, while phosphacan was diffusely expressed in the stratum oriens and radiatum of Ammons horn, and in the hilus and inner one-third molecular layer of the dentate gyrus at the 2nd month after birth. There was no difference in the expression of neurocan and phosphacan between IER and SD rats at the 2nd month after birth. By contrast, phosphacan was reduced in the inner molecular layer of the dentate gyrus in 8-month-old IER, while neurocan was reexpressed in the outer molecular layer and hilus in 3- and 8-month-old IER. It was suggested that the insufficient expression of neurocan may affect the development of neuronal organization in the hippocampus, and that the remodeling of extracellular matrix in the dentate gyrus may contribute to the mossy fiber sprouting into the inner molecular layer.

Immunohistochemical localization of histamine N-methyltransferase in guinea pig tissues.

Histamine plays important roles in gastric acid secretion, inflammation, and allergic response. Histamine N-methyltransferase (HMT; EC 2.1.1.8) is crucial to the inactivation of histamine in tissues. In this study we investigated the immunohistochemical localization of this enzyme in guinea pig tissues using a rabbit polyclonal antibody against bovine HMT. The specificity of the antibody for guinea pig HMT was confirmed by Western blotting and the lack of any staining using antiserum preabsorbed with purified HMT. There was strong HMT-like immunoreactivity (HMT-LI) in the epithelial cells in the gastrointestinal tract, especially in the gastric body, duodenum, and jejunum. The columnar epithelium in the gallbladder was also strongly positive. Almost all the myenteric plexus from the stomach to the colon was stained whereas the submucous plexus was not. Other strongly immunoreactive cells included the ciliated cells in the trachea and the transitional epithelium of the bladder. Intermediately immunoreactive cells included islets of Langerhans, epidermal cells of the skin, alveolar cells in the lung, urinary tubules in the kidney, and epithelium of semiferous tubules. HMT-LI was present in specific structures in the guinea pig tissues. The widespread distribution of HMT-LI suggests that histamine has several roles in different tissues.

Purification and characterization of alkaline phosphatase from Bacillus stearothermophilus.

Soluble alkaline phosphatase from the thermophilic bacterium Bacillus stearothermophilus was purified by a combination of chromatographic methods, and its properties were examined. The purified enzyme had specific activity of 4.43 μmol p‐nitrophenol/min per mg of protein and seemed to be a single band on SDS/PAGE with a molecular mass of 32 kDa. Its apparent Km for p‐nitrophenyl phosphate was 1.114 mM. The enzyme exhibited an optimal pH of approx. 9.0 and exhibited its highest activity at 60–70 °C. It also showed a bivalent cation requirement for activity, with maximal enhancement in the presence of Mg2+. In addition, significant thermal stability was observed in comparison with counterparts from mesophiles. Its partial N‐terminal sequence was T1FSIVAFDPATGELGIAVQ19 as estimated by automated Edman degradation method. A search on the SwissProt database did not reveal any similar protein sequences from other sources.

Developmentally regulated expression of brain-specific chondroitin sulfate proteoglycans, neurocan and phosphacan, in the postnatal rat hippocampus.

Abstract. Developmental changes in the distribution of brain-specific chondroitin sulfate proteoglycans, neurocan and phosphacan/RPTPζ/β, in the hippocampus of the Sprague-Dawley rat were examined using monoclonal antibodies 1G2 and 6B4. The 1G2 immunoreactivity was predominant in the neonatal hippocampus while the 6B4 immunoreactivity was predominant in the mature hippocampus. Moderate 1G2 immunoreactivity was detected in the dentate gyrus and subiculum immediately after birth. Immunoreactivity reached a peak on postnatal days 7–10 (P7–P10) when intense 1G2 labeling was present throughout the neuropil layers of the hippocampus except the mossy fiber tract. 6B4 immunoreactivity was limited in the stratum lacunosum moleculare of CA1 in the neonatal hippocampus. It gradually increased by P21 when diffuse 6B4 immunoreactivity was detected in the stratum oriens and radiatum of Ammons horn, and in the hilus and inner one-third molecular layer of the dentate gyrus, while 1G2 immunoreactivity decreased after P21. In the adult hippocampus, moderate 6B4 immunoreactivity was present in the stratum oriens and radiatum of Ammons horn, and in the hilus and inner one-third molecular layer of the dentate gyrus, but not in the mossy fiber tract. In addition, strong 6B4 labeling appeared around a subset of neurons after P21. The results suggest that neurocan may have a role in the development of neuronal organization, while phosphacan/RPTPζ/β may contribute to the maintenance and plasticity of synaptic structure and function. Furthermore, the absence of 1G2 and 6B4 immunoreactivities in the stratum lucidum suggests that neurocan and phosphacan/RPTPζ/β may function as a barrier for the extension of mossy fibers and provide an environment permissive for fasciculation of the mossy fibers.

Immunohistochemical localization of histamine N-methyltransferase in the bovine central nervous system

Dopamine is the principal neurotransmitter that modulates locomotion, emotion, memory and learning, and neuroendocrine functions in the mammalian CNS. To clarify the role of dopamine signaling, we have generated dopamine-deficient mice by a transgenic rescue approach. At juvenile stage, dopamine-deficient mice displayed motor dysfunctions that were characterized by a marked decrease in locomotor activity, blockade of methamphethamineinduced hyperactivity, and cataleptic behavior. These mutant mice also had a severe defect in active avoidance learning with a shuttle box paradigm. On the other hand, development and function of the pituitary gland were normally maintained in spite of the reduced dopamine metabolism. Our results demonstrate that dopamine is essential for motor and emotional control during postnatal development.