Ryujiro Shoji

Developmental change in the glycosaminoglycan composition of the rat brain.

Abstract: Glycosaminoglycans (GAGs) were isolated from the brains of pre‐ and postnatal rats. The GAG content of the brain, based on the amount of DNA, was constant during the period from day 13 to day 15 of gestation. After day 15, the GAG content began to increase and reached a plateau by 10 days after birth. Hyaluronate (HA) was the main GAG (> 60% of the total) in the fetal rat brain, and the relative amount of HA decreased after birth. Conversely, the relative amount of chondroitin sulfate increased with development and reached the adult level by 20 days after birth. Heparan sulfate (HS) was the major sulfated GAG in the fetal rat brain at early developmental stages, but HS accounted for approximately 10% of the total GAG in the postnatal brains. In addition to these GAGs, a polysialosyl glycoconjugate was isolated from rapidly growing brains of the rat. These three GAGs could be isolated either from the cerebellum, cerebrum, or brainstem of the newborn rat. A closely similar age‐related change in the GAG composition was observed in each of these different regions of the brain. The developmental change could be implicated in morphogenesis or maturation of the brain.

Changes with Development in the Expression of Cathepsin E in the Fetal Rat Stomach

The changes with development in the expression of cathepsin E in the fetal rat stomach were examined immunochemically and immunohistochemically. The activity of acid proteinase in fetal gastric extracts increased dramatically during late gestational stages, rising from 0.017 units per mg of protein on day 15 of gestation to 0.591 units per mg of protein on day 21 of gestation. Electrophoretic analysis, combined with immunological tests, showed that the increase was due exclusively to increases in the activity of the monomeric and dimeric forms of cathepsin E, while SDS‐PAGE‐immunoblot analysis revealed that both forms are present as a 43‐kDa proenzyme. Immunohistochemically, cathepsin E was localized in the cytoplasm of all proliferating epithelial cells of pars glandularis on day 16 of gestation or later. As revealed by conventional histological methods, surface mucous cells and parietal cells appeared for the first time in specimens on day 19 of gestation, and all of these cells were immunopositive for cathepsin E. The present study further indicated that cathepsin E is the predominant aspartic proteinase in the stomach of young rats, until pepsinogen C appears. Based on these results, possible roles of gastric cathepsin E are discussed.

Vestibulocochlear Defects and Effects of Deuterium Oxide in Mutant Bustling (BUS) Mice

Bustling mouse (BUS/Idr: bus) is a mutant mouse strain which exhibits bustling/hyperkinetic behavior and functional disorders related to the vestibulocochlear system such as rapid circling and loss of auditory startling response. In homozygous (bus/bus) mice this phenotype develops progressively after birth and has been shown from cross experiments to be inherited by a single autosomal recessive gene. Using light and electron microscopy, we examined the development of pathological changes in the inner ear of homozygous mice. Effects of deuterium oxide, which has been shown to influence vestibular function, on the abnormal behavior of homozygous mice of different ages were also examined. Pathological changes of the inner ear including impairment and/or loss of auditory hair cells, deformation and/or loss of the inner and outer tunnels of the organ of Corti, hypoplasia of the otoliths, and decrease in number of neurons of the spiral genglion were observed in the homozygous mice starting before the weaning stage and progressively thereafter, but not in the heterozygous mice. Although histological changes were also noted in the crista ampullaris and maculae, they were less evident than those in the cochlea at least until the mice were 6 weeks old. Administration of deuterated physiological saline (8-16 ml/kg, per os) tended to decrease the abnormal behavior of the homozygous mice, including rapid circling. These morphological and functional findings suggest that BUS mice may be a useful model for analysis of the pathogenesis of vestibulo-cochlear disorders.

BUS/ldr, a Mutant Mouse Strain Exhibiting Abnormal Behaviors: Behavioral Similarities of BUS Mice and Chemically Labyrinthectomized Mice

The behavioral abnormalities of homozygotes (bus/bus) of mutant bustling mice, BUS/ldr, were characterized in comparison with those of heterozygotes (+/bus) and of chemically labyrinthectomized mice. Homozygotes were 4‐fold more active than heterozygotes, as evaluated in an open field (11 × 17 cm) for 24 hr by means of Animex. Homozygotes revealed the lack of righting reflex and of head nystagmus on a rotating board, the frequent backward‐moving and the inability to swim, in addition to characteristic behaviors such as circling, head bobbing and head tossing. Bilaterally labyrinthectomized mice behaviorally mimicked BUS homozygotes: The behavioral patterns of operated animals were essentially the same as those of BUS homozygotes with respect to all reflex and locomotor indices employed, suggesting that some peripheral vestibular dysfunction, though unidentified yet, is attributed to abnormal behaviors of BUS mice. Unilaterally labyrinthectomized mice were not hyperactive, nor exhibited circling behavior.

Mammalian Embryo Culture from the Pre-Implantation Stage, and Analysis of Developmental Failures due to Chromosomal Abnormalities in Vitro*

Abstract In vitro culture methods make it possible to manipulate mammalian embryos experimentally, and continuous observation of the developmental processes furnishes significant information for mammalian embryology. The advancement in the culture methods of the mouse embryos from the pre‐implantation through the limb bud stage is reported here, along with a description of one of the applied experiments of embryo culture from the pre‐implantation stage in the Chinese hamster.

Three distinct molecular species of proteoglycan synthesized by the rat limb bud at the prechondrogenic stage

To characterize proteoglycans in the prechondrogenic limb bud, proteoglycans were extracted with 4 M guanidine HCl containing a detergent and protease inhibitors from Day 13 fetal rat limb buds which had been labeled with [35S]sulfate for 3 h in vitro. About 90% of 35S-labeled proteoglycans was solubilized under the conditions used. The proteoglycan preparation was separated by DEAE-Sephacel column chromatography into three peaks; peak I eluted at 0.45 M NaCl concentration, peak II at 0.52 M, and peak III at 1.4 M. Peaks I and III were identified as proteoglycans bearing heparan sulfate side chains. The heparan sulfate proteoglycan in peak III was larger in hydrodynamic size than the proteoglycan in peak I. The heparan sulfate side chains of peak III proteoglycan were smaller in the size and more abundant in N-sulfated glucosamine than those of peak I proteoglycan. Peak II contained a chondroitin sulfate proteoglycan with a core protein of a doublet of Mr 550,000 and 500,000. The chondroitin sulfate proteoglycan was easily solubilized with a physiological salt solution and the heparan sulfate proteoglycan in peak I was partially solubilized with the physiological salt solution. The remainder of the proteoglycan in peak I and the heparan sulfate proteoglycan in peak III could be solubilized effectively only with a solution containing a detergent, such as nonanoyl-N-methylglucamide. This observation indicates the difference in the localization among these three proteoglycans in the developing rat limb bud.

Structure and Functions of Cell Surface Glycoconjugates in Brain Morphogenesis

Abstract Migration of newly‐generated neuroblasts to their definitive locations to form the laminated architecture of the brain is crucial in brain morphogenesis. In order to understand the molecular mechanism operative in the neuroblast migration, cell surface or pericellular glycoconjugates were isolated from the rat brains at various developmental stages. Hyaluronate (HA), chondroitin sulfate proteoglycans (CS‐PGs), heparan sulfate proteoglycans (HS‐PGs) and polysialosyl glycoproteins (N‐CAMs) have been characterized so far. Our biochemical and immunohistochemical experiments indicate that HA and a CS‐PG participate in construction of the hydrated extracellular milieu for the directional migration of neuroblasts.

Developmental change in the amount of polysialosyl glycopeptides isolated from the rat brain.

Abstract: Polysialosyl glycopeptides were coisolated with glycosaminoglycans by Pronase digestion of the whole brains of perinatal rats and could be separated from known glycosaminoglycans by two‐dimensional electrophoresis on cellulose acetate film. The polysialosyl glycopeptides could not be obtained from fetal rat brain on day 13 of gestation, but began to be detected on day 14. The amount of polysialosyl glycopeptides was estimated from the dye concentration of the Alcian blue‐stained spot in the electrophoreto‐gram. The glycopeptide content increased almost linearly, on the basis of brain DNA, up to 10 days after birth. Thereafter, the content decreased rapidly, and hardly any polysialosyl glycopeptides could be isolated from the brain at ∼30 days. This developmental change may be involved in morphogenesis and maturation of the brain. The polysialosyl glycopeptides could be isolated from the cerebellum, from the cerebrum, or from the brainstem of the neonatal rat. However, each region of the brain had a postnatal developmental change in glycopeptide content different from those of the other regions.