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Previous studies have shown that cellular prion protein (PrPC) plays anti‐apoptotic and anti‐oxidative role against cell death induced by serum‐deprivation (SDP) in an immortalized prion protein gene‐deficient neuronal cell line derived from Rikn prion protein (PrP) gene‐deficient (Prnp–/–) mice, which ectopically produce excess Doppel (Dpl) (PrP‐like glycoprotein). To investigate whether PrPCinhibits apoptotic neuronal cell death without Dpl, an immortalized cell line was established from the brain of ZrchI Prnp–/–mice, which do not show ectopic expression of Dpl. The results using a ZrchI neuronal Prnp–/–cell line (NpL2) showed that PrPCpotently inhibited SDP‐induced apoptotic cell death. Furthermore, PrPCexpression enhanced the superoxide dismutase (SOD) activity in NpL2 cells. These results indicate that Dpl production did not affect anti‐apoptotic and anti‐oxidative functions of PrP, suggesting that PrPCmay be directly correlated with protection against oxidative stress.

Emx1 and Emx2, mouse orthologs of the Drosophila head gap gene, ems, are expressed during corticogenesis. Emx2 null mutants exhibit mild defects in cortical lamination. Segregation of differentiating neurons from proliferative cells is normal for the most part, however, reelin-positive Cajal-Retzius cells are lost by the late embryonic period. Additionally, late-born cortical plate neurons display abnormal position. These types of lamination defects are subtle in the Emx1 mutant cortex. In the present study we show that Emx1 and Emx2 double mutant neocortex is much more severely affected. Thickness of the cerebral wall was diminished with the decrease in cell number. Bromodeoxyuridine uptake in the germinal zone was nearly normal; moreover, no apparent increase in cell death or tetraploid cell number was observed. However, tangential migration of cells from the ganglionic eminence into the neocortex was greatly inhibited. The wild-type ganglionic eminence cells transplanted into Emx1/2-double mutant telencephalon did not move to the cortex. MAP2-positive neuronal bodies and RC2-positive radial glial cells emerged normally, but the laminar structure subsequently formed was completely abnormal. Furthermore, both corticofugal and corticopetal fibers were predominantly absent in the cortex. Most importantly, neither Cajal-Retzius cells nor subplate neurons were found throughout E11.5-E18.5. Thus, this investigation suggests that laminar organization in the cortex or the production of Cajal-Retzius cells and subplate neurons is interrelated to the tangential movement of cells from the ganglionic eminence under the control of Emx1 and Emx2.

Formation of highly organized neocortical structure depends on the production and correct placement of the appropriate number and types of neurons. POU homeodomain proteins Brn-1 and Brn-2 are coexpressed in the developing neocortex, both in the late precursor cells and in the migrating neurons. Here we show that double disruption of both Brn-1 and Brn-2 genes in mice leads to abnormal formation of the neocortex with dramatically reduced production of layer IV-II neurons and defective migration of neurons unable to express mDab1. These data indicate that Brn-1 and Brn-2 share roles in the production and positioning of neocortical neuron development.

We generated transgenic mice carrying enhanced green fluorescent protein (EGFP) under the control of the nestinsecond-intronic enhancer (E/nestin:EGFP). Flow cytometry followed by in vitroassays revealed that in situEGFP expression in the embryonic brain correlated with the mitotic index, the cogeneration of both neurons and glia, and the frequency of neurosphere formation in vitro.High-level EGFP expressors derived from embryos included a distinct subpopulation of cells that were self-renewable and multipotent, criteria that define neural stem cells (NSCs). Such cells were largely absent among lower-level or non-EGFP expressors, thereby permitting us to enrich for NSCs using EGFP expression level. In adults, although E/nestin:EGFP-positive cells included the NSC population, the frequency of neurosphere formation did not correlate directly with the level of EGFP expression. However, moderately EGFP-expressing cells in adults gained EGFP intensity when they formed neurospheres, suggesting embryonic and adult NSCs exist in different microenvironments in vivo.

The pressure-flow relationship of erythrocyte suspension in the bullfrog’s hind limb was studied by the perfusion method in which the rate of flow: pressure characteristic consists of a smooth curve convex to the pressure axis, tending to a straight line at a high rate of flow. This asymptotic line was found to have a negative intercept on the flow axis, which is independent of the haematocrit of the sample, and depended only on the radius of the vascular tube. In the light of the marginal zone theory, our finding appears appropriate.

Summary As in our previous report (Kamino, Uyesaka & Inouye,J. Membrane Biol.17:13, 1974), the absorbance changes of murexide caused by Ca2+ and followed up by a dual wavelength spectrophotometer were applied to measure synaptosomal Ca2+-binding in the presence of cations such as Rb+, Mn2+ or La3+. All the cations tested showed a significant inhibition of synaptosomal Ca2+-binding except Li+. The inhibitory effects could be divided into the following three categories: (1) noncompetitive, co-operative K+-type, which includes alkali metal ions. The potency of inhibition is K+>Rb+>Cs+>Li+, Na+=0; (2) competitive Mn2+-type which includes many divalent cations. The inhibitory potency was found to be in the following order: Mn2+≫Sr2+>Cd2+, Ba2+>Mg2+; (3) nonspecific, noncompetitive La3+-type: among the cations tested, La3+ and Ce3+ were found to markedly reduce the Ca-binding capacity of synaptosomal particles, resulting in a noncompetitive inhibition, at least in the range of Ca2+ concentration used.

Some characteristics and the degree of intestinal absorption in the developing human fetus were examined by measuring solute evoked potentials and 14C-D-glucose uptake into the everted jejunal segments.In all segments, the Michaelis-Menten relationship was observed between D-glucose concentrations and the evoked potentials or D-glucose uptake. Increase of Na-ion concentrations enhanced both D-glucose evoked potentials and uptake.Both D-glucose and L-α-alanine evoked potentials increased in a significant correlation to the fetal age; however, the apparent Michaelis constants did not show any significant change. The structural specificity of sugar for generating evoked potentials was the same as that reported for adult mammals. Among amino acids, only the L-form of neutral and acidic amino acids generated markedly high evoked potentials, but the basic ones hardly at all. Oligopeptides such as glycyl-glycine and glycyl-glycyl-glycine also generated high evoked potentials.

Summary Using nitroxide fatty acid spin labels, the effects of some cations such as La3+, Cd2+ and Hg2+ on synaptosomal membranes were studied by observing changes in their ESR spectra. The labels were incorporated almost instantaneously into synaptosomes isolated from rat brain cortex. ESR spectra of the spin-labeled synaptosomes were significantly broadened immediately upon adding La3+, Ce3+, Cd2+ or Hg2+ but hardly affected by Ca2+, Sr2+ and Ba2+. The magnitude of the change in the separation of the outer two peaks in ESR spectra (2T?') depends on the number (n) of methylene units between the polar head group and the spin-label (nitroxide) group; that is, it increases with decreasingn. Among these ions, the effect of La3+ was the greatest and appeared to be in parallel with the amount of La3+ bound with the synaptosomes. On the other hand, K+, Rb+ or Li+ causes hardly any significant changes.

Summary Ruthenium red combines with isolated synaptosomes, resulting in strong inhibition of their Ca2+-binding. In isotonic saline media, however, the dye-induced inhibition of Ca2+-binding is significantly greater than that expected for the amount of bound dye and Hill's exponent of the Ca2+-binding decreases to 1 with an increase in the amount of the dye bound. On the other hand in isotonic mannitol-sucrose solution, inhibition of synaptosomal Ca2+-binding brought about by the dye is proportional to the amount of dye bound. Based on these results, the effects of the dye on the co-operative nature of synaptosomal Ca2+-binding is discussed.

Summary Free ion concentration of some divalent heavy metal ions such as Mn2+, Co2+, Ni2+, Cd2+ and Zn2+ in the synaptosomal suspension was measured to determine binding with synaptosomes isolated from rat brain cortex. A dual wavelength spectrophotometer was utilized to monitor the absorbance changes of murexide raised by stepwise addition of these ions (as chloride salts). Such titration experiments of the synaptosomal suspension revealed that a part of the added divalent cation such as Mn2+, Co2+ or Ni2+ was almost instantaneously bound to synaptosomes in isotonic NaCl media. Our previous study (Kamino, Uyesaka & Inouye,J. Membrane Biol.17:13, 1974) demonstrated that raised external K+ resulted in a specific noncompetitive inhibition of synaptosomal Ca-binding. Just like the Ca-binding, Mn-, Co- or Ni-binding was almost completely depressed by high external K+ or ruthenium red when the free concentration of the cations was 10 µm or less, while at higher concentrations the binding was not affected. The present results indicate that tested divalent cations bind with both “Ca-binding sites” and “non-Ca-binding sites” of synaptosomal membrane, the nature of the binding sites of both being quite different: the former is sensitive to high external K+ and to ruthenium red but the latter is not.

Through screening of a murine brain cDNA library, we have isolated two brain specific cDNAs encoding highly homologous proteins, named 921-L and 921-S, comprised of 134 amino acids with 80% identity. Immunohistological study with the mAbs raised against the bacterially expressed 921 proteins showed that 921-L protein is distributed at the dendritic region and 921-S at the neuronal somatic surface. Immuno-electron microscopic study revealed that both 921 proteins are localized at the presynaptic terminal, indicating that the 921 proteins are differentially expressed at the dendritic and somatic presynapses.

There is increasing interest in the role of RNA-binding proteins during neural development.DrosophilaMusashi is one of the neural RNA-binding proteins essential for neural development and required for asymmetric cell divisions in theDrosophilaadult sensory organ development. Here, a novel mammalian neural RNA-binding protein, mouse-Musashi-1, was identified based on the homology toDrosophilaMusashi andXenopusNRP-1. In the developing CNS, mouse-Musashi-1 protein was highly enriched in the CNS stem cell. Single-cell culture experiments indicated that mouse-Musashi-1 expression is associated with neural precursor cells that are capable of generating neurons and glia. In contrast, in fully differentiated neuronal and glial cells mouse-Musashi-1 expression is lost. This expression pattern of mouse-Musashi-1 is complementary to that of another mammalian neural RNA-binding protein, Hu (a mammalian homologue of aDrosophilaneuronal RNA-binding protein Elav), that is expressed in postmitotic neurons within the CNS.In vitrostudies indicated that mouse-Musashi-1 possesses binding preferences on poly(G) RNA homopolymer, whereas Hu is known to preferentially bind to short A/U-rich regions in RNA. Based on their differential expression patterns and distinct preferential target RNA sequences, we believe that the mouse-Musashi-1 and Hu proteins may play distinct roles in neurogenesis, either through sequential regulatory mechanisms or differential sorting of mRNA populations during asymmetric division of neural precursor cells.

It has been reported that prenatal X-irradiation of rats during the late gestation period causes heterotopic Purkinje cells in the internal granular layer (IGL) of the abnormally foliated cerebellum. The present study was designed to demonstrate the process of X-ray-induced derangement of Purkinje cells and their surrounding cells. In addition, the expression of some morphoregulatory molecules was examined to determine which molecules are involved in the abnormal pattern of Purkinje cells. Pregnant rats (n = 22) were exposed to 2.5 Gy X-radiation on gestation day 21 and the cerebellum of progeny was examined histologically and by immunohistochemistry to identify Purkinje and Bergmann cells. At 12 h after exposure, extensive cell death was observed in the external granular layer (EGL). By postnatal day (P) 9, while Purkinje cells with well-developed dendrites aligned underneath the EGL in the control cerebellum, Purkinje cells with shorter and abnormally oriented dendrites failed to align and remained in the heterotopic location in the IGL. Bergmann cells and their fibers were also disoriented but later recovered in their proper position. Abnormal folia developed in the irradiated rats. Using immunohistochemistry, we next examined the levels of the neural cell adhesion molecule (NCAM), fibronectin, tenascin, and Reelin. Among them, only the level of Reelin was affected significantly. Reelin decreased strikingly in the premigratory zone of the EGL and IGL in the irradiated cerebellum on P1, and the decrease continued until P9. Decreased Reelin expression was demonstrated quantitatively by Northern blot analysis and the correlation between the mRNA and protein levels was well presented. The expression of reelin mRNA decreased significantly by irradiation from P0, being almost one third of the level in controls on P4, and tended to recover up to P9. It is thus indicated that X-irradiation causes a marked decrease in the level of Reelin at the critical stage for the alignment of Purkinje cells. Since Reelin has been shown to play an important role in the migration of neural cells, it is suggested that the decrease in Reelin by X-irradiation is an important factor for the derangement of Purkinje cells.

A rapid-access control method for optical disk drives has been developed. A brake pulse is fed forward to the objective lens actuator to rapidly reduce track crossing velocity of the light spot when in the close vicinity of the destination track during macro seeking. At the instant the light spot reaches the destination track, the track-following operation is performed by only the objective lens actuator until the linear motor stops. This rapid-access system was applied to a 3.5" optical disk drive, and it was found to provide a seek time for 256 tracks, excluding rotational latency, 30% shorter than a conventional direct access system.

Fast data retrieval of the optical disk memory can be attributed to fast head accessing along with stable tracking and focusing servos and high speed decoding of error correcting code. To realize fast head access, an access control technique, with accurate position and speed information obtained from the disk surface and state observer, has been developed. A digitized servo circuit achieved a stable pull on the destination track after seek motion. By the above technique, less than 70 msec of the average access time was attained in a "full-height" sized optical disk drive. To realize high speed decoding, an error detection and correction processor with applied Eucl-idian algorithm has been developed. This paper describes the details of technology used to achieve the fast data retrieval.

In order to create a rapid accessing feature of optical disk drives, the optical head should be positioned as accurately as possible near the target track if seeking motion is desired. First, an accurate seeking span decision algorithm employed to predict the spiral tracks' rotational number during seeking is discussed. Second, a method of correcting a miscount in the number of crossing tracks for accurate track-count accessing is proposed. During the seeking operation, the method of correcting the track miscount can generate a virtual track-crossing pulse in real time when a track-crossing pulse is missed due to a distorted tracking error signal during seeking. These two methods are practically installed in a 130 mm International Organization for Standardization (ISO) format optical disk drive and effectively operated for accurate track-count accessing.

The requirement for an optical disk drive to operate properly under the existence of violent external vibration has been growing because portability of the drive increases as it becomes miniaturized. In order to improve a servo system's resistance to external vibration, an objective lens velocity feedback method in a two-stage tracking servo system and two-degree-of-freedom velocity control drive of a linear motor during the seeking operation are proposed. These methods are incorporated in 90 mm optical disk drives, and a vibration test is performed to investigate improvements in the servo performance. When the vibration is 0.5 gat 50 Hz, the average seeking time increases only 10% when compared with the condition of no external vibration.