Factors Contributing to the Poor Myelination in the Brain of the Snell Dwarf Mouse

Abstract: Conventional histological examination of the pituitary does not distinguish Snell dwarf mutants (dw/dw) from their normal littermates (+/?) in the neonatal stage. However, immunohistochemical examination of pituitaries of litters born to heterozygous Snell parents revealed that in approximately 25% of the glands examined, the number of positive cells was very low in the neonatal stage. We attempted to delineate the events resulting in the poor myelination in the brain of the Snell dwarf mouse, and to devise an immunohistochemical method for identifying the mutant neonate. Differences in the brain weights of the dw/dw and +/? mice first became apparent on the 10th day of age, and from this time on no further increase in the weight of the dwarf mouse brain was recorded. Increase in CNPase activity was found to be suppressed in the cerebrum and brain stem throughout the developmental stage, but not in the other parts of the brain. The yield of isolated myelin decreased by 58% in the mutant mouse, but CNPase activity was equivalent to that of control myelin. Differences in DNA content per cerebrum from the dw/dw and +/? mice first became apparent on the 10th day of age. Henceforth, the dw/dw mice showed no further increase, although the +/? mice continued to increase. [³H]Thymidine incorporation into the DNA fraction in vivo on the 7th day of age, when glial cell proliferation in the cerebrum is most active, was suppressed to about 50% of the control level in all parts of the dwarf brain. These findings indicate that the poor myelination found in the mutant cerebrum is a hypomyelination due to reduced oligodendroglial proliferation caused by lack of circulating growth hormone.     

Neurochemical Studies on the Cerebellar Hypoplasia of Gunn Rat (Hereditary Hyperbilirubinemic Rat)

The cerebellar hypoplasia induced by hereditary hyperbilirubinemia in the Gunn rat was analyzed neurochemically and immunohistochemically. The antiserum against myelin basic protein was used to visualize the arborization of the fibers in the cerebellum. Arborization was very scarce in the affected lobes of the homozygous (jj) cerebellum. Na,K-ATPase activity did not show significant differences between the jj and the control (Jj) cerebellum. The concentration of norepinephrine in the jj cerebellum was about 1.5 times that of the control. However, the activation ratio of the Na,K-ATPase by norepinephrine and other catecholamines such as dopamine and isoproterenol was about twice as high as the basal activity, and no significant difference was observed between the jj and the Jj cerebella. The glutamic acid decarboxylase activity of the jj cerebellum did not differ significantly from that of the control.     

Neurochemical Characteristics of Myelin-like Structure in the Chick Retina

Abstract: Certain characteristics of myelin-like structures in the chick retina were examined morphologically and biochemically. Developmental changes of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in the chick retina and optic nerve were examined. The measurable activity in the retina was first detected at 16 days of incubation and thereafter, it increased rapidly until 4 weeks post-hatching. By contrast, CNPase activity in the optic nerve reached the maximum level at 4 days post-hatching and maintained a constant level thereafter. The purifed myelin fraction from the chick retina showed higher activity of CNPase, whereas its activity in the retinal homogenate was very low. Hence, it was considered that the myelin fraction from the chick retina is similar to that of CNS myelin with respect to CNPase. Protein profiles of the purified myelin fractions isolated from the chick optic tectum, optic nerve, retina and sciatic nerve were analysed by SDS-polyacrylamide gel elec-trophoresis. Myelin fractions from the chick optic tectum and optic nerve contained basic protein (BP) and Folch-Lees proteolipid protein (PLP). Myelin fraction from the chick sciatic nerve contained BP, P2 and two glycoproteins (PO and 23K). In contrast, retinal myelin fraction contained only BP. PLP, PO, 23K and P2 proteins were definitely undetectable. Electron micrographs revealed that some axons in the optic nerve fiber layer of the chick retina were wrapped by a spiral-structured myelin-like sheath, which showed some differences from those of CNS and PNS myelin sheaths. It was suggested that the origin of the myelin-like structure in the chick retina is other than from oligodendroglia or Schwann cells.     

Morphological and Biochemical Studies on the Cerebral Cortex from Reeler Mutant Mice: Development of Cortical Layers and Metabolic Mapping by the Deoxyglucose Method

Abstract: Cerebral cortex from reeler mutant mice was examined morphologically and biochemically. The sequential process of postnatal cell migration in the cerebral cortex of reeler (rl/rl) was examined morphologically. The dense cellular cortical plate lies below the molecular layer near the cerebral surface just after birth in normal mice while in reeler most of the cells are concentrated in the center of the cortex. In the cortex of adult reeler, the broad laminar structure of the neurons could be seen to form inverted positions in the cortical layers. The total wet weight, and the concentration of DNA and RNA in the pallium cerebri from reeler did not differ significantly from those in the control. As to the protein profiles of the pallium cerebri detected by SDS- polyacrylamide gel electrophoresis, no significant differences were observed. Activities of CNPase (2',3'-cyclic nucleotide 3'-phosphohydrolase), which is a myelin enzyme of CNS, and choline acetyltransferase were at the same level in both the reeler and the control. Therefore, reeler mutation does not appear to affect the genetically determined cell numbers, number of cholinergic fibers, and myelination. By autoradiographic observation of the cerebral cortex after intraperitoneal injection of [¹⁴C]2-deoxyglucose, it was revealed that 2-deoxyglucose was incorporated intensively into the fourth layer (granular layer) of the cerebrum from the control. In reeler it was also incorporated into the granular layer but in a more widespread distribution. We conclude that terminals to the granular layer make metabolically active synapse, perhaps even in a manner inverted from normal.     

Intramolecular and intermolecular perturbation on electronic state of FAD free in solution and bound to flavoproteins: FTIR spectroscopic study by using the C = O stretching vibrations as probes

The intramolecular and intermolecular perturbation on the electronic state of FAD was investigated by FTIR spectroscopy by using the C=O stretching vibrations as probes in D(2)O solution. Natural and artificial FADs, i.e. 8-CN-, 8-Cl-, 8-H-, 8-OCH(3)-, and 8-NH(2)-FAD labelled by 2-(13)C, (18)O=C(2), or 4,10a-(13)C(2) were used for band assignments. The C(2)=O and C(4)=O stretching vibrations of oxidized FAD were shifted systematically by the substitution at the 8-position, i.e. the stronger the electron-donating ability (NH(2) > OCH(3) > CH(3) > H > Cl > CN) of the substituent, the lower the wavenumber region where both the C(2)=O and C(4)=O bands appear. In contrast, the C(4)=O band of anionic reduced FAD scarcely shifted. The 1,645-cm(-1) band containing C(2)=O stretching vibration shifted to 1,630 cm(-1) in the medium-chain acyl-CoA dehydrogenase (MCAD)-bound state, which can be explained by hydrogen bonds at C(2)=O of the flavin ring. The band was observed at 1,607 cm(-1) in the complex of MCAD with 3-thiaoctanoyl-CoA. The 23 cm(-1) shift was explained by the charge-transfer interaction between oxidized flavin and the anionic acyl-CoA. In the case of electron-transferring flavoprotein, two bands associated with the C(4)=O stretching vibration were obtained at 1,712 and 1,686 cm(-1), providing evidence for the multiple conformations of the protein.     

Engineering the substrate specificity of porcine kidney D-amino acid oxidase by mutagenesis of the "active-site lid"

Comparison of the primary structures of pig kidney D-amino acid oxidase (DAO) and human brain D-aspartate oxidase (DDO) revealed a notable difference at I215-N225 of DAO and the corresponding region, R216-G220, of DDO. A DAO mutant, in which I215-N225 is substituted by R216-G220 of DDO, showed D-aspartate-oxidizing activity that wild-type DAO does not exhibit, together with a considerable decrease in activity toward D-alanine. These findings indicate that I215-N225 of DAO contributes profoundly to its substrate specificity. Based on these results and the crystal structure of DAO, we systematically mutated the E220-Y224 region within the short stretch in question and obtained five mutants (220D224G, 221D224G, 222D224G, 223D224G, and 224D), in each of which an aspartate residue is mutated to E220-Y224. All of the mutants exhibited decreased apparent K(m) values toward D-arginine, i.e., to one-seventh to one-half that of wild type DAO. The specificity constant, k(cat app)/K(m app), for D-arginine increased by one order of magnitude for the 221D224G or 222D224G mutant, whereas that for D-alanine or D-serine decreased to marginal or nil.     

Identification of the C=O stretching vibrations of FMN and peptide backbone by 13C-labeling of the LOV2 domain of Adiantum phytochrome3

Phototropin, a blue-light photoreceptor in plants, has two FMN-binding domains named LOV1 and LOV2. We previously observed temperature-dependent FTIR spectral changes in the C=O stretching region (amide-I vibrational region of the peptide backbone) for the LOV2 domain of Adiantum phytochrome3 (phy3-LOV2), suggesting progressive structural changes in the protein moiety (Iwata, T., Nozaki, D., Tokutomi, S., Kagawa, T., Wada, M., and Kandori, H. (2003) Biochemistry 42, 8183-8191). Because FMN also possesses two C=O groups, in this article, we aimed at assigning C=O stretching vibrations of the FMN and protein by using 13C-labeling. We assigned the C(4)=O and C(2)=O stretching vibrations of FMN by using [4,10a-13C2] and [2-13C] FMNs, respectively, whereas C=O stretching vibrations of amide-I were assigned by using 13C-labeling of protein. We found that both C(4)=O and C(2)=O stretching vibrations shift to higher frequencies upon the formation of S390 at 77-295 K, suggesting that the hydrogen bonds of the C=O groups are weakened by adduct formation. Adduct formation presumably relocates the FMN chromophore apart from its hydrogen-bonding donors. Temperature-dependent amide-I bands are unequivocally assigned by separating the chromophore bands. The hydrogen bond of the peptide backbone in the loop region is weakened upon S390 formation at low temperatures, while being strengthened at room temperature. The hydrogen bond of the peptide backbone in the alpha-helix is weakened regardless of temperature. On the other hand, structural perturbation of the beta-sheet is observed only at room temperature, where the hydrogen bond is strengthened. Light-signal transduction by phy3-LOV2 must be achieved by the progressive protein structural changes initiated by the adduct formation of the FMN.     

Effects of hydrogen bonds in association with flavin and substrate in flavoenzyme d-amino acid oxidase. The catalytic and structural roles of Gly313 and Thr317.

According to the three-dimensional structure of a porcine kidney D-amino acid oxidase-substrate (D-leucine) complex model, the G313 backbone carbonyl recognizes the substrate amino group by hydrogen bonding and the side-chain hydroxyl of T317 forms a hydrogen bond with C(2)=O of the flavin moiety of FAD [Miura et al. (1997) J. Biochem. 122, 825-833]. We have designed and expressed the G313A and T317A mutants and compared their enzymatic and spectroscopic properties with those of the wild type. The G313A mutant shows decreased activities to various D-amino acids, but the pattern of substrate specificity is different from that of the wild type. The results imply that the hydrogen bond between the G313 backbone carbonyl and the substrate amino group plays important roles in substrate recognition and in defining the substrate specificity of D-amino acid oxidase. The T317A mutant shows a decreased affinity for FAD. The steady-state kinetic measurements indicate diminished activities of T317A to substrate D-amino acids. The transient kinetic parameters measured by stopped-flow spectroscopy revealed that T317 plays key roles in stabilizing the purple intermediate, a requisite intermediate in the oxidative half-reaction, and in enhancing the release of the product from the active site, thereby optimizing the overall catalytic process of D-amino acid oxidase.     

Isolation and characterization of phytase isozymes produced by Aspergillus oryzae

A mutant strain (KL-38) of Aspergillus oryzae was obtained by UV irradiation. Phytase activity of KL-38 in molded rice (koji rice) was about 2.7-fold of that obtained from the parent strain (BP-1). Phytase activity of KL-38 in the submerged culture was similar to that of BP-1. Two types of phytase were produced from koji culture: phytase I (Phy I) was produced during incubation of both koji and submerged cultures, and phytase II (Phy II) was obtained only from koji culture. Phy II production was increased in KL-38 compared with BP-1, whereas the production of Phy I was similar for both KL-38 and BP-1. This finding indicates that A. oryzae has at least two types of phytase isozyme.