Masaharu Miyake

Developmental Changes of N-Acetyl-L-Aspartic Acid, N-Acetyl-α-Aspartylglutamic Acid and β-Citryl-L-Glutamic Acid in Different Brain Regions and Spinal Cords of Rat and Guinea Pig

Abstract The developmental changes of N‐acetylaspartic acid (NA‐Asp), N‐acetyl‐α‐aspartylglutamic acid (NA‐Asp‐Glu), and β‐citryl‐L‐glutamic acid (β‐CG) have been examined in the cerebrum, cerebellum, brain stem and spinal cord of both rat and guinea pig by the gas chromatographic method developed in our studies. A rapid increase in the concentration of NA‐Asp was observed postnatally in every region of the rat brain. On the other hand, all regions of guinea pig brain showed the prenatal increases. NA‐Asp‐Glu showed a different developmental profile, depending on region of the brain, in the two species. The concentration of NA‐Asp‐Glu remained constantly low during brain maturation in the rostral regions. In the caudal portions it showed a marked increase during maturation and reached a high level in the adult brain. The concentration of β‐CG was highest at birth in all regions of rat brain and rapidly decreased by 20 days after birth and remained low thereafter. The rapid decrease occurred in the guinea pig during the foetal period, and β‐CG content decreased to an adult level at birth.

Sox6 overexpression causes cellular aggregation and the neuronal differentiation of P19 embryonic carcinoma cells in the absence of retinoic acid

The Sox6 gene is a member of the Sox gene family that encodes transcription factors. Previous studies have suggested that Sox6 plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia and fibroblast‐like cells. In this report, we have shown that Sox6 mRNA increased rapidly in P19 cells during RA induction and then decreased during the differentiation of P19 into neuronal cells. To explore the possible roles of Sox6 during this process, stably Sox6‐overexpressing P19 cell lines (P19[Sox6]) were established. These P19[Sox6] had acquired both characteristics of the wild‐type P19 induced by RA. First, P19[Sox6] cells showed a marked cellular aggregation in the absence of RA. Second, P19[Sox6] could differentiate into microtubule‐associated protein 2 (MAP2)‐expressing neuronal cells in the absence of RA. Sox6 expression could cause the activation of endogenous genes including the neuronal transcription factor Mash‐1, the neuronal development‐related gene Wnt‐1, the neuron‐specific cell adhesion molecule N‐cadherin, and the neuron‐specific protein MAP2, resulting in neurogenesis. Moreover, E‐cadherin, a major cell adhesion molecule of wild‐type P19, was strongly induced by Sox6, resulting in cellular aggregation without RA. Thus Sox6 may play a critical role in cellular aggregation and neuronal differentiation of P19 cells.

Suppression of Sox6 in P19 cells leads to failure of neuronal differentiation by retinoic acid and induces retinoic acid-dependent apoptosis.

The Sox6 gene is a member of the Sox gene family, which encodes transcription factors, and previous studies have suggested that it plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia, and fibroblast‐like cells. Sox6 mRNA increases rapidly in P19 cells during RA induction and then decreases during differentiation into neuronal cells. To investigate whether Sox6 expression is essential for neuronal differentiation, we established Sox6‐suppressed P19 (P19[anti‐Sox6]) cells by transfection of antisense‐Sox6 cDNA. Most of the P19[anti‐Sox6] cells showed no neurites and were not stained by the anti‐MAP 2 antibody, while the suppression of Sox6 expression nearly totally blocked neuronal differentiation in P19 cells. Further, Sox6 suppression caused RA‐dependent apoptosis by P19[anti‐Sox6] cells: RA‐treated P19[anti‐Sox6] cells showed chromatin condensation, DNA fragmentation, and an increase in caspase‐3‐like activity. Thus, Sox6 is considered essential for neuronal differentiation and may play an important role in the early stages of neuronal differentiation or apoptosis.

Polyethylene glycol modification of interleukin-6 enhances its thrombopoietic activity

Abstract This study was conducted to increase the in vivo thrombopoietic activity of interleukin-6 (IL-6). Recombinant human IL-6 was covalently conjugated with N -succinimidyl succinate monomethoxy polyethylene glycol (PEG). The in vitro bioactivity of the PEG-modified IL-6 was reduced with increase in its degree of PEG modification, but the in vivo thrombopoietic activity of PEG-modified IL-6 was markedly increased compared to unmodified IL-6. In particular, modified IL-6, in which 54% of the 14 lysine amino groups were coupled with PEG, showed > 10 times greater thrombopoietic effect in vivo than unmodified IL-6. The area under the serum concentration curve of PEG-modified IL-6 after subcutaneous injection was > 17 times larger than that of unmodified IL-6. Chemical attachment of PEG to IL-6 thus increased the bioavailability of IL-6, and may facilitate its potential therapeutic use.

Isolation and identification of β-citryl-L-glutamic acid from newborn rat brain

Abstract Ab unknwon compound containing glutamic acid residue was found in newborn rat brain. The compound occurred predominantly in brain. Its concentration was approx. 1 μmol/g tissue at birth and decreased to one-tenth 24 days after birth. The compound was isolated from newborn rat brains, and subjected to elementary analysis and to infrared and mass spectrometric analysis. Glutamic acid and citric acid were formed from the compound on acid hydrolysis. The compound was presumed to be a citryglutamic acid. Two isomers, α- and β-citrylglutamic acid, were sunthesized. The unknown compound was identified as β-citryl- L -glutamic acid. The occurrence of this compound has not been reported in nature.

Synthesis and degradation of methylated proteins of mouse organs: Correlation with protein synthesis and degradation

L-(Methyl-14C)-methionine was administered i.p. to mice, and the incorporation of radioactive methionine into proteins and methyllysine and methylarginine residues formed by the transfer of the methyl-14C group of methionine were measured. Tissue protein was actively methylated in organs having a high activity of protein synthesis, and the in vivo methylating activity in organs was not correlated with theprotein methylating activity of the organs determined in vitro. Puromycin inhibited both protein synthesis and protein methylation in mouse organs to a similar degree. Neither the formation of S-adenosyl-(methyl-14C)-methionine nor protein methylase was inhibited by puromycin. The data suggests that proteins are methylated immediately after protein synthesis, that is, newly synthesized proteins are the substrates of protein methylation. Radioactive methionine and the [C14] methyl groups of methyllysine and methylarginine residues of tissue proteins are degraded in parallel over a period of 3 wk, suggesting that protein methylation is an irreversible type of protein modification.

Purification and Properties of Calmodulin‐Lysine N‐Methyltransferase from Rat Brain Cytosol

Abstract: A S‐adenosylmethionineiprotein‐lysine N‐methyltransferase (EC 2.1.1.43) has been purified from rat brain cytosol 7,080‐fold with a yield of 8%, using octopus calmodulin as a substrate. It contains a lysine residue that is not fully methylated. The enzyme was purified by ammonium sulfate fractionation, Sephacryl S‐200 gel filtration, and phosphocellulose and octopus calmodulin‐Sepharose affinity chromatographies. Among protein substrates, it was highly specific toward octupus calmodulin. The Km values for octopus calmodulin and S‐adenosyl‐L‐methionine were found to be 2.2 × 10−8M and 0.8 × 10−6M, respectively. The molecular weight was estimated to be 57,000 by gel filtration and the pH optimum was between 7.5 and 8.5. The enzyme was stimulated in the presence of 10−7M Mn2+ and 10−4M Ca2+. HPLC of the acid hydrolysate of methyl‐3H‐labeled calmodulin showed the formation of ε‐N‐mono‐, ε‐N‐di‐, and ε‐N‐trimethyllysine. Reverse‐phase HPLC of tryptic peptides of the methyl‐3H‐labeled calmodulin demonstrated that the labeled N‐methyllysine lies in the 107–126 peptide. These findings suggest that this enzyme methylated a specific lysine residue of octopus calmodulin.

Expression of kininogen mRNAs and plasma kallikrein mRNA by cultured neurons, astrocytes and meningeal cells in the rat brain

Expression of kininogen mRNAs has been studied in cultures of three different types of cells in rat brain, including neurons and astrocytes from cerebral cortex and meningeal cells from the leptomeninges/choroid plexus. T-kininogen mRNA was expressed by meningeal cells, but not by neurons and astrocytes, and the expression in meningeal cells was enhanced by culture with prostaglandin E2 (PGE2) or dibutyryl cAMP (Bt2cAMP). Low-molecular-weight kininogen mRNA was not detected in these cultures of cells, even after treatment with PGE2. Although expression of high-molecular-weight kininogen mRNA was very low in these cultures of cells, PGE2 or Bt2cAMP markedly stimulated its expression in cultures of meningeal cells and slightly in neurons, but not in astrocytes. We also found that expression of plasma kallikrein mRNA was strong in cultures of meningeal cells and slight in astrocytes, but absent in neurons. These results suggest that cells in the leptomeninges/choroid plexus are major sources of kininogens in rat brain which may function as precursor proteins for kinins and/or potent cysteine proteinase inhibitors during cerebral inflammation.

A rapid and sensitive method for the determination of hypusine in proteins and its distribution and developmental changes

A simple and sensitive method for determining hypusine in proteins was developed. A greater part of amino acids in the acid hydrolysate of proteins was separated from hypusine by treatment with an ion-exchange resin. The sample containing partially purified hypusine was then analyzed by high-performance liquid chromatography using the post-column derivatization method with o-phthalaldehyde. The recovery rate of hypusine through the overall procedure was more than 95%. Using this method, the distribution and developmental changes of hypusine in proteins were determined. The amino acid was found in proteins of all examined organs of rat. Its concentration was 5-40 nmol/g protein. The subcellular distribution in rat liver was also determined. About 60% of total amount of hypusine was present in the proteins of cytoplasmic and microsomal fractions and its relative concentration was high in the proteins of microsome and lysosome and low in mitochondria. In developing rat, the concentration of hypusine in the brain proteins was relatively high during the first 2 or 3 weeks of postnatal life and then decreased until adulthood. Its concentration in the liver proteins was highest at birth and then decreased continuously to the adult level.

Correlation of the level of β-citryl-l-glutamic acid with spermatogenesis in rat testes

beta-Citryl-L-glutamic acid, which is known to be highly concentrated in the brains of immature animals, is preferentially localized in the testes of various adult animals, including mammals, amphibians and fish, mainly in the germinal cells. In young rats, the citrylglutamate concentration increases with age and coincides with the development of late spermatocytes into early spermatids. Rats with seminiferous tubule failure induced by ductuli efferentes ligation and experimental cryptorchidism are infertile as a result of germ cell depletion, especially spermatocytes and early spermatids. In these animals, the testicular citrylglutamate content was much lower than in normal testes.