Strain differences to effects of aging on concentrations of amino acids in cerebrospinal fluid between Sprague Dawley rat and Wistar Kyoto rat.

Age-related alterations and differences of weights and those of amino acid concentrations in the cerebrospinal fluid (CSF) were evaluated between Sprague Dawley (SD) rats and Wistar Kyoto (WKY) rats from eight to twenty weeks of age. The weights of SD rats were heavier than WKY rats at all ages. The age-related alterations of the CSF concentration of many amino acids within each strain were significant but showed no significant trend with age. Between the strains, the concentration differences of excitatory and inhibitory amino acids were not frequent although the concentrations of arginine, alanine and threonine were significantly higher in SD rats than in WKY rats. These results suggest that the different CSF concentrations of amino acids may relate to characteristics of rat strains.     

Purification and Characterization of a Ca2+-Dependent Protein Kinase from the Halotolerant Green Alga Dunaliella tertiolecta

A Ca²⁺-dependent protein kinase (CDPK) that has been partiallypurified and characterized previously [Yuasa and Muto (1992)Arch. Biochem. Biophys. 296: 175] was further purified to about20,000-fold from the soluble fraction of Dunaliella tertiolecta.The enzyme preparation contained 60- and 52-kDa polypeptidesboth of which phosphorylated casein as a substrate. Both polypeptidesshowed a Ca²⁺-dependent increase in mobility during SDS-PAGEand ⁴⁵Ca²⁺-binding activity after SDS-PAGE and electroblottingonto a nitrocellulose membrane, suggesting that both the 60-and 52-kDa CDPKs directly bind Ca²⁺. The protein kinase inhibitors,K-252a and staurosporine, inhibited the CDPK competitively withrespect to ATP. An antibody raised against the 60-kDa CDPK crossreactedwith both the 60- and 52-kDa polypeptides. Both molecular specieswere autophosphorylated in the presence of Ca²⁺, and a highlyphosphorylated 80-kDa band appeared in addition to these phosphorylatedbands at 60 and 52 kDa in SDS-PAGE. However, the specific activityof CDPK was not changed by prior autophosphorylation when theautophosphorylated enzyme was assayed as a mixture of thesephosphorylated molecular species. Only the 60-kDa polypeptidewas immunodetected in subcellular fractions of Dunaliella cells.The 52-kDa polypeptide increased during storage of the enzyme.These results suggest that the 52-kDa polypeptide is a proteolyticartifact produced during purification. Immunoreactive bandsof 60-kDa were detected in extracts of several green algae butnot in extracts of higher plants or a brown alga. ¹This research was partly supported by Grants-in-Aid from theMinistry of Education, Science and Culture, Japan (No. 06454013and 06304023) and Research Fellowship of the Japan Society forthe Promotion of Science for Young Sciencists. ²Research Fellow (PD) of the Japan Society for the Promotionof Science.     

Kinetics and collagenolytic role of eosinophils in chronic gastric ulcer in the rat

 An association between eosinophils and tissue damage has been observed in numerous disorders. However, few reports have addressed the role of infiltrating eosinophils in gastric ulcer healing. The aim of this study was to investigate the kinetics and role of eosinophils infiltrating experimental chronic gastric ulcers in the rat. We developed a monoclonal antibody against human matrix metalloproteinase 1 (MMP1) purified from conditioned culture medium of human skin fibroblasts. Acetic acid-induced gastric ulcers were resected from rats on days 1, 3, 5, 10, 20, 40, and 180 after the days of induction (day 0). Tissue specimens were immunostained with this antibody and examined with an electron microscope. Few eosinophils were observed in the granulation tissue until day 20. By days 40 and 180, MMP1-positive eosinophils had increased in the granulation tissue of open ulcers. Azan staining revealed dispersed collagen fibers around infiltrating eosinophils. In contrast, scars demonstrated few eosinophils in fibrous tissue on days 40 and 180. Eosinophils which express MMP1 infiltrate granulation tissue at the chronic stage of gastric ulceration. The results suggest that eosinophils may play a role in tissue remodeling and deterioration of ulceration. Accepted: 18 March 1997     

Differential Localization of the γ3 and γ12 Subunits of G Proteins in the Mammalian Brain

Abstract: The localization of two forms of the γ subunit of G proteins, γ₃ and γ₁₂, was examined in the mammalian brain. Concentrations of these two γ subunits increased markedly, as did those of glial fibrillary acidic protein, during postnatal development in the rat cerebral cortex. In aged human brains, by contrast, the concentration of γ₃ tended to decrease with age, whereas that of γ₁₂ in the temporal cortex increased slightly. An immunohistochemical study of human brains revealed that γ₃ was abundant in the neuropil, whereas γ₁₂ was localized in glial cells. In the hippocampal formation of aged human brains, levels of γ₁₂-positive cells, as well as levels of glial fibrillary acidic protein- and vimentin-positive astrocytes, increased, in particular in the CA1 subfield and the prosubiculum, in which there was a decrease in the number of pyramidal cells. The appearance of γ₁₂-positive cells associated with the loss of pyramidal cells was also observed in the hippocampus of rats that had been treated with kainic acid. These results indicate that γ₁₂ is strongly expressed in reactive astrocytes. In a study of cultured neural cells, we found that γ₁₂ was predominant in glioma cells, such as C6 and GA-1 cells, in contrast with the specific localization of γ₃ in PC12 pheochromocytoma cells, which are neuron-like cells. Taken together, the results indicate that γ₃ and γ₁₂ are selectively expressed in neuronal and glial cells, respectively, and that concentrations of γ₃ and γ₁₂ in the brain are related to the numbers and/or extent of maturation of these cells.     

Subunit constitution of carbonic anhydrase from Chlamydomonas reinhardtii

Carbonic anhydrase purified from the cell surface of Chlamydomonas reinhardtii was inactivated by treatment with dithiothreitol. This treatment caused dissociation of the holoenzyme into 35-kDa (A) and 4-kDa (B) subunits as revealed by SDS/PAGE. The 35-kDa subunit was further separated into two components A1 (35 kDa) and A2 (36.5 kDa) by SDS/PAGE using a gradient gel. These two components have the same amino acid sequence up to at least the 10th amino acid from the N-terminus. The molecular masses were estimated at 76 kDa and 35 kDa for the holoenzyme and the large subunit, respectively, and the molar ratio of the former to the latter at 1:2, by using the techniques of low-angle laser light-scattering photometry and precision differential refractometry combined with gel-filtration HPLC. The molar ratio of the 35-kDa/4-kDa subunits was estimated at 1:1 the gel-filtration HPLC monitored with precision differential refractometry. Atomic-absorption spectrophotometry revealed that the holoenzyme contains two atoms of zinc. These results suggest that the holoenzyme is a heterotetramer composed of two large subunits (A1 and A2) and two small subunits (B).     

Possible role of rat fatty acid-binding proteins in the intestine as carriers of phenol and phthalate derivatives

Fatty acid-binding proteins of hepatic and intestinal type and gastrotropin-like protein (GTLP) were purified from rat intestinal cytosol by Sephadex G-75 gel filtration and DEAE-cellulose, CM-cellulose, and hydroxylapatite chromatographies. In addition to fatty acids, butylated hydroxytoluene (BHT), phthalate dibutyl, and di(2-ethylhexyl) esters (DBP and DEHP) were identified by gas liquid chromatography and mass spectrometry as endogenous ligands from the extract of either fatty acid-binding protein superfamily. These protein families in the intestine may have an important role as carriers in the initial step of arresting these exogenous pollutants.     

Identification of lactotropes (PRL cells), somatotropes (GH cells), corticotropes (ACTH cells) and thyrotropes (TSH cells) in the pituitary gland of the musk shrew, Suncus murinus L. (Insectivora), by immunohistochemistry

By means of immunohistochemistry, lactotropes, somatotropes, corticotropes and thyrotropes in the pituitary of the male musk shrew, Suncus murinus L., were identified at the electron-microscopic level. Lactotropes were classified into three types: type I containing large (200-450 nm in diameter) round secretory granules, type II with medium-sized (150-250 nm in diameter) ones and type III with small (50-150 nm in diameter) ones. Somatotropes were also classified into type I somatotropes that contain large (450 nm in diameter) spherical secretory granules and type II somatotropes containing comparatively small (300 nm in diameter) round granules. Both type I and II somatotropes were small and sometimes contained rod-shaped granules. Corticotropes were round or oval cells with round secretory granules in various densities and sizes (150-500 nm in diameter) scattered all over the cytoplasm. Thyrotropes were angular or polyhedral cells containing electron-transparent round secretory granules (200-300 nm in diameter) and large irregularly shaped granules with a maximum diameter of about 1,500 nm. Each type of the cells may be distinguished by its respective ultrastructural characteristics alone without the aid of immunohistochemistry.