Ca2(+)-calmodulin-dependent protein kinase II phosphorylates various types of non-epithelial intermediate filament proteins

We have investigated the actions of Ca2(+)-calmodulin (CaM)-dependent protein kinase II on various types of non-epithelial intermediate filament proteins, vimentin, desmin, glial fibrillary acidic protein (GFAP) and neurofilament triplet proteins. Most of these filament proteins could serve as substrates. The effects of phosphorylation on the filamentous structure of vimentin were investigated in sedimentation experiments and by using electron microscopy. The amount of unassembled vimentin increased linearly with increased phosphorylation. However, the extent of the effect of phosphorylation on the potential to polymerize was also affected by the MgCl2 concentration, under conditions for reassembly. The actions of Ca2(+)-CaM-dependent protein kinase II on non-epithelial intermediate filaments under physiological conditions are given attention.     

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.     

Amphotericin B resistance is recessive in Chinese hamster hybrid cells

Previous studies from our laboratory have shown that Chinese hamster V79 cells mutated to high level resistance to amphotericin B have a lower cellular level of cholesterol, the target molecule for the polyene antibiotic. Two amphotericin B-resistant (AMB^R) mutants were each hybridized to their parental amphotericin B-sensitive (AMB^S) V79 cells. All the hybrids derived from AMB^R/AMB^S fusions were as sensitive to polyene antibiotics (amphotericin B, filipin, and pimaricin) as AMB^S/AMB^S hybrids. The AMB^R/AMB^S hybrids were found to contain cholesterol per phospholipids that is comparable to those in AMB^S or AMB^S/AMB^S. The analysis of hybrids formed between mutant and wild-type cells thus indicated that resistance to amphotericin B is a recessive marker, and that the cellular level of cholesterol is compensated in the AMB^S/AMB^R hybrids. Hybrids of AMB^R and AMB^R cells were all resistant, so that the three AMB^R mutants all fell into a single complementation group.     

2-Deoxyglucose Incorporation in the Cerebellum of Weaver and Nervous Mutant Mice

Abstract: The 2-deoxyglucose autoradiographic method has been used to study activity in cerebellum of the weaver and nervous mutant mice. Patterns of 2-deoxyglucose incorporation into the cerebral hemispheres from weaver and nervous strains did not differ significantly from those of the controls. In the normal cerebellum, 2-deoxyglucose incorporation was maximal in the granular layer, where mossy fibers form synapses with the dendrites of granule cells. In the cerebellum of nervous mice, which lacks Purkinje cells, the incorporation of the 2-deoxyglucose was maximal in the granular layer, but the incorporation into the molecular layer appeared less than in the control. The incorporation into the cerebellum from weaver, which lacks granule cells, was much higher than that of the control, the maximal incorporation being found in the Purkinje cell layer and in cell masses located in the white matter. These data suggest that the heterologous synapses that mossy fibers or climbing fibers form with the cells in the Purkinje cell layer and the cells in the white matter in the weaver cerebellum are functional.     

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.     

Cerebro-costo-mandibular syndrome

Summary A 7-month-old boy with the cerebro-costomandibular syndrome is presented. This is the first case report in an Oriental population.15 reported cases in the literature are reviewed.     

Continuous culture of reuber hepatoma cells in serum-free,arginine-, glutamine-and tyrosine-deprived chemically defined medium

Summary The rat hepatoma cell line, H4-II-E, was grown serially over a I-year period and about 30 passages in arginine-, glutamine-, and tyrosine-deprived and ornithine-supplemented Eagle's mininum essential medium with no supplements other than biotin. The adapted cel line, R-Y121B, proliferates in the above mentioned medium with a doubling time of about 4 days and maintains hepatic “marker” enzymes such as tyrosine aminotransferase, phenylalanine hydroxylase, and all the enzymes of the urea cycle. This work was supported in part by Grant-in-Aid for Cancer Research 301050 and Science Research Grant 337013 from the Ministry of Education, Science and Culture, Japan.     

Identification of a protease inhibitor from rat peritoneal macrophages as poly(ADP-ribose)

Rat peritoneal macrophages contain a chymotrypsin-like protease and its specific inhibitor, both being associated with chromatin of the cells. The inhibitor was separated from the protease by gel filtration through a Sephadex G-75 column, further treated with trypsin, DNase and RNase, and then purified successively on Sephadex G-75, Sephadex G-25, and dihydroxyboryl Bio-Gel P-60 columns. The purified inhibitor had a molecular weight in the range from 2,000 to 3,500 and an absorption maximum at 260 nm at pH 7.0. When the inhibitor was digested by snake venom phosphodiesterase, the inhibitory potency was lost, yielding 5'-AMP and 2'-(5'-phosphoribosyl)-5'-AMP as the digestion products which were identified by high pressure liquid chromatography. The inhibitory potency was neutralized specifically by anti-poly(ADP-ribose) antiserum. The profile of inhibition by the isolated inhibitor was nearly identical with that produced by authentic poly(ADP-ribose). It was therefore concluded that the inhibitor isolated was identical with poly(ADP-ribose), whose chain length ranged from 4 to 7 ADP-ribosyl units. This is the first demonstration that a intracellular protease inhibitor can be endogenous poly(ADP-ribose).