Shusuke Hirano

Purifications and properties of basic proteins in pig spinal cord and peripheral nerve.

Abstract 1. 1. Two different basic proteins were purified, one from pig spinal cord and the other from pig peripheral nerve, utilizing the methods of (a) acetone treatment, (b) 0.03 M HCl extraction at pH 2.0, (c) pH 7.0 precipitation and (d) CM-cellulose and (e) Sephadex G-75 column chromatography. The elution pattern of acid extract from pig spinal cord on the CM-cellulose column showed a major peak, eluted with approx. 0.43 M NaCl, which was further purified on a Sephadex G-75 column and the main peak, eluted at 1.5 of v e / v 0 , was obtained. On the other hand, extract from pig peripheral nerve showed two peaks, cluted with approx. 0.27 M and 0.43 M NaCl on a CM-cellulose column. The first peak was submitted to chromatography on Sephadex G-75 and another basic protein, eluted at 2.2 of v e / v 0 , was purified. 2. 2. On the disc gel electrophoresis of Reisfeld et al. 8 , the purified proteins from pig spinal cord and peripheral nerve showed a single band of R F 0.60 and 1.07, respectively, relative to lysozyme. By ultracentrifugal analysis, each showed a single symmetrical peak. 3. 3. The ultraviolet absorption pattern and the amino acid composition of a purified basic protein from pig peripheral nerve were quite different from those of the protein from pig spinal cord, the characteristics of which were closely related to those of an experimental allergic encephalitogenic protein already described by other workers.

Changes in ganglioside composition and morphological features during the development of cultured astrocytes from rat brain

Changes in ganglioside content over a period of days were examined in astrocytes obtained via cell passage from rat cerebral cortex. Thin-layer chromatography revealed that, in the astrocytes, ganglioside GM1 was absent, the predominant ganglioside being GM3. Also, an increased GD3 content in long-term astrocyte cultures was detected. The morphological features of astrocytes were also studied using immunoperoxidase staining. Astroglial features were characterized by high levels of glial fibrillary acidic protein (GFAP) and vimentin, which are the major intermediate-filament proteins present in astrocytes at an early culture stage. In long-term-cultured (greater than 7 months) astrocytes, vimentin and GFAP were increased in process-bearing cells. Ganglioside GD3 recognized by R24 monoclonal antibody was also expressed in these cells. These results suggest that the increase of ganglioside GD3 in long-term-cultured astrocytes may be related to the appearance of multistellate cells showing strong reactivity against GFAP and vimentin during development over a specified period in culture.

Characteristics of primary cultured neurons from embryonic mutant El mouse cerebral cortex

To elucidate the differences between neurons of epileptogenic animals and those of normal animals, cellular characteristics of neurons of mutant strain El mice which are highly susceptible to seizures were investigated using immunocytochemical techniques. In neurons of 3-day primary cultures, the control ddY mouse neurons showed dividing stages in about 0.2% of neurofilament (NF)-positive neurons, whereas no dividing neurons were observed among the NF-positive El mouse neurons. In 7-day cultures, localization of GD3 ganglioside in the proliferating control ddY mouse neurons was observed, but there was no GD3 ganglioside in the mutant El mouse neuron. The content of GD3 ganglioside detected by high-performance thin-layer chromatography of El mouse cultured cells was ca. 1/4 of that of ddy mice. These findings suggest that neurons of the El mouse are differentiated earlier than those of the control ddY mouse.

Developmental change in ganglioside expression in primary culture of rat neurons

Developmental changes in ganglioside levels and patterns were investigated in neuronal cells dissociated from 17-day-old fetal rat hemispheres for up to 7 days of culture. Increases in ganglioside contents and the onset of GM3 synthesis, which is associated with proliferation of glial cells, were observed as the neuronal network was established in cell cultures. The distribution of gangliosides in developing neurons was monitored by the indirect immunofluorescent technique using three anti-ganglioside antibodies. Anti-GM1 antibody showed immunofluorescence only on the cell soma 1 and 3 days after plating and additional binding between cell aggregates by 7 days in culture. GD3 ganglioside, the predominant species in embryonic neurons, was not detected on the neuronal cell surface, whereas the number of positively stained non-neuronal cells was increased at 7 days. Monoclonal A2B5 antibody suggested that polysialogangliosides play a role in neuronal network formation. In 1-day-old culture, however, all antibodies bound poorly to cell surface antigens and strongly to cells, the membranes of which were permeabilized with acetone. These results suggest that a substantial amount of gangliosides are retained, transformed within the cell to more complex gangliosides, and translocated to the cell surface following neurite outgrowth and morphological changes.

A simple infrared spectroscopic method for the measurement of expired 13CO2.

Abstract For the analysis of 13CO2 and 12CO2 in exhaled breath the mass spectrometer has been employed in general, but it is not convenient for clinical use and maintenance. We have been successfully performing the continuous measurement of expired 13CO2 and 12CO2 with a new analyzer using infrared spectroscopy which is easy to manipulate and maintain. This analyzer measures 12CO2 in a short cell with an absorbancy of around 2360.2 cm−1 and 13CO2 in a long cell with an absorbancy of around 2272.0 cm−1, recording the volume percentage of 12CO2 and the atom percentage excess of 13CO2. In our experiment, using a normal male rat weighing 210 g, a maximum expired 13CO2 of 1.7 atom% excess was recorded 10 min after an intraperitoneal injection of 10 mg of [13C]-sodium bicarbonate containing 49.0 atom% of the isotope.

Differences in ME-LI and VIP-LI in discrete brain regions of seizure-naive and seizure-experienced El mice

In an attempt to elucidate the relationship between endogenous methionine-enkephalin (ME) and vasoactive intestinal polypeptide (VIP) with generalized seizures, we determined regional brain levels of ME-like and VIP-like immunoreactivity (ME-LI and VIP-LI) in El mice during and after seizures induced by repeated tossing stimulation. The levels of ME-LI in the striatum and hippocampus of seizure-naive El mice (El−) were lower than those of the control ddY mice, the mother strain of El mice. Conversely, the level of VIP-LI in the medulla oblongata and pons of El− was higher than that of ddY mice. The level of ME-LI in the striatum of seizure-experienced El mice (El+) killed 96 hours after three consecutive seizures was high, while levels of VIP-LI in the striatum and hypothalamus were low, in comparison to those of El− mice. A detailed time-course study revealed that seizures in El mice caused (1) significant decreases in levels of ME-LI in the striatum and hippocampus during seizures, (2) a significant decrease of VIP-LI content in the striatum 3 hours after seizures, and (3) a significant increase in hypothalamic VIP-LI 9 hours after seizures. These observations suggest that ME and VIP may play some role in El mouse seizures.

Effects of hyperphenylalaninemia in the fetal stage on the postnatal development of fetal rat brain.

The effect of exposure at different prenatal stages to maternal hyperphenylalaninemia (HyPhe) on the somatic and neurological development of fetuses in rats was studied, with special respect to the change of relevant enzyme activities in the brain. While evident somatic damage was found only in the fetuses exposed to maternal HyPhe at a last stage of gestation, distinct mental retardation seemingly due to some irreversible damage to the brain was observed in all the treated fetuses regardless of the timing of exposure, and a significantly reduced activity of 2′, 3′-cyclic nucleotide 3′-phosphohydrolase (CNPase), a marker enzyme of myelin, was confirmed in the mantle region of the brain.

Preparation of astrocyte-deficient cultures containing neurons and oligodendroglia from embryonic rat cerebral hemispheres

Complement-dependent anti-glial fibrillary acidic protein (GFAP) antibody-mediated cytotoxicity was utilized in order to prepare astrocyte-deficient cultures from mechanically dissociated cells from 18-day-old embryonic rat cerebral hemispheres. Neurons and a lesser number of oligodendroglia were the major cell components in such cultures.

Age-dependent changes in monkey lenticular gangliosides

The content, composition, and distribution of gangliosides were examined in the lenses of normal rhesus monkeys aged 6-16 years. Gangliosides were isolated by organic solvent extraction. DEAE-Sephadex ion-exchange column chromatography, and thin-layer chromatography (TLC). Ganglioside contents determined by the thiobarbituric acid method increased in the lens with aging. TLC analysis of gangliosides showed a much more complex pattern with aging, and the predominant gangliosides were tentatively identified as GM3, GM1, and GD1a. Individual lenticular gangliosides were identified by TLC-immunostaining procedures using anti-GM1 and anti-asialoGM1 antisera.

Effect of Colchicine on Ganglioside Composition of Rat Primary Culture Neurons

Developmental changes in gangliosides in the course of neurite outgrowth were examined in dissociated fetal rat cerebral neurons in culture. About a 2-fold increase in ganglioside levels was seen with the progression of neurite formation for up to 24 h in predominantly neuronal cultures. Ganglioside patterns appeared to be unchanged during the first 24 h, subsequently consisted of higher amounts of GD3 and b-series gangliosides (such as GD1b, GT1b, and GQ1b), and lower amounts of a-series gangliosides (GM1 and GD1a). Although the addition of colchicine to the cell growth medium inhibited neurite outgrowth in developing neurons, little if any differences in ganglioside patterns were found between control and colchicine-treated cells. Ganglioside levels decreased slightly in colchicine-treated cells in agreement with the decrease in cell attachment to culture dishes. Although colchicine treatment 8 h after plating caused complete retraction of formed neurites, the ganglioside level of the cells continued to increase during the following 16-hour incubation. Thus, the data suggest that ganglioside synthesis in differentiating neurons does not primarily accompany the expansion in cell surfaces due to neurite formation, and raises the possibility that a large proportion of gangliosides is retained in intracellular compartments.