Tetsuo Nakabayashi

Higher avidity binding of apolipoprotein (E–AII) complex than of apolipoprotein E monomer to β‐amyloid

Apolipoprotein E (apoE) is believed to be closely involved in the pathogenesis of Alzheimers disease (AD) because of its ability to bind to β‐amyloid (Aβ), the primary component of senile plaques. The presence of cystein residues in apoE2 and apoE3 allows these isoforms to form disulfide‐linked complexes, such as apo(E–AII) complex and apo(AII–E–AII) complex. A 50‐kDa complex [which corresponded to apo(E–AII)–Aβ, because it reacted with any of the three antibodies, anti‐apoE, anti‐apoAII, or anti‐Aβ] was detected by immunoblot analysis in native cerebrospinal fluid (CSF) obtained from nondementia patients with the apoE phenotype E3/E3. However, a band considered to represent apoE–Aβ was not observed. The dissociation constant (Kd) values obtained for the specific binding of recombinant apoE2, apoE3, and apoE4 to Aβ1–42 were 48.1 ± 2.2 nM, 63.7 ± 2.1 nM, and 75.9 ± 1.8 nM, respectively. In contrast, the binding affinity of the partially purified apo(E3–AII) complex to Aβ1–42 was very high, the Kd being 5.5 ± 0.5 nM. No basic difference was observed between lipidated and nonlipidated apoE in terms of the characteristics of the binding of apoE isoforms to Aβ1–42; however, lipidation reduced the binding capacity of each isoform in a dose‐dependent manner. These findings seem consistent with the generally accepted idea that apoE4 is a risk factor for AD, insofar as only apoE4 is unable to form a complex with apoAII owing to its lack of a cystein residue. In addition, it is possible that apoE3 monomer (and possibly apoE2 monomer), like apoE4 but unlike apo(E–AII) complex, can act as a risk factor in the pathogenesis of AD. J. Neurosci. Res. 58:301–307, 1999.

Evaluation of the automatic fluorescent image analyzer, Image Titer, for quantitative analysis of antinuclear antibodies.

By making comparisons with the usual manual method, we evaluated an automatic fluorescent image analyzer (Image Titer, Tripath Imaging, Burlington NC), the software for which was developed to simplify measuring indirect immunofluorescent antinuclear antibodies (FANAs). In this new system, images of the stained sample are displayed, and it measures the FANA titer and staining pattern using only 1 slide per subject and does not required the staining of a series of diluted samples as does the manual method. This system showed good reproducibility and linearity for 4 types of control serum samples (with homogeneous, speckled, discrete speckled, and nucleolar staining patterns). In 132 serum samples, consistency between the methods was 100% for the FANA staining pattern and 93.9% for the FANA titer. The Image Titer system detected each pattern in samples with 2 mixed patterns. This system should partly reduce labor and lead to results with minimum differences among individuals, including newly trained persons.

Stressful delivery influences circulating thrombopoietin (TPO) levels in newborns: possible role for cortisol in TPO-mpl binding

The regulation mechanism of circulating thrombopoietin (TPO) level in human newborns remains unknown. In the present study, we examined whether the TPO concentrations in cord blood were influenced by the difference in the delivery method and the presence or absence of maternal/fetal complications. Cortisol levels were simultaneously measured to assess the adrenal response of fetuses. Both the TPO level and the cortisol level were substantially greater in the neonates delivered vaginally with and without the complications than in those delivered by cesarean section without the complications. The binding assay showed that the incubation of mpl(+)/BaF3 cells with cortisol gave rise to a significant decrease in the binding sites of TPO. These results suggest that the stress to the fetuses near the time of delivery affects the cord blood TPO levels, which may be mediated in part by the action of cortisol on the TPO-mpl binding system.

Effect of apolipoprotein AII on the interaction of apolipoprotein E with ?-amyloid: Some apo(E-AII) complexes inhibit the internalization of ?-amyloid in cultures of neuroblastoma cells

Apolipoprotein (apo) E and its polymorphism are linked to the pathogenesis of late‐onset and sporadic Alzheimers disease (AD). ApoE facilitates the deposition and fibrillogenesis of β‐amyloid (Aβ), and may participate in Aβ clearance. We recently found that apo(E–AII) complex binds to Aβ much more strongly than does monomeric apoE. Here, we investigated the effect of apoAII on the interaction between apoE and Aβ. Addition of apoAII to apoE monomers increased the binding of apoE2 and apoE3 to Aβ1–42, presumably following the formation of apo(E3–AII), apo(E2–AII), and apo(AII–E2–AII) complexes. This increased binding was not seen in the case of apoE4. When neuroblastoma cells were cultured in media containing Aβ1–42 and a mixture of apoE3 and apoAII, intracellular Aβ was significantly reduced and cell viability was maintained at a higher level than in cells cultured without apoAII. ApoE2 itself seemed to act as an inhibitor of the endocytosis of Aβ, and we did not observe a significant effect of apoAII on the movement of Aβ in apoE2‐containing medium. However, cell viability could be maintained at a higher level (as with apoE3) by adding apoAII to apoE2, despite the reduced viability of cells incubated without apoAII. In medium containing apoE4, both the amount of Aβ accumulated into cells and the cell viability were unchanged by the presence of apoAII in the medium. In addition, apoE4 itself was toxic, as previously suggested. These findings demonstrate that the type of apo(E–AII) complex present could underlie the isoform‐specific role of apoE in the pathogenesis of AD. J. Neurosci. Res. 62:608–614, 2000.

Analysis of hemoglobin and globin chain variants by a commonly used capillary isoelectric focusing method

To analyze both hemoglobin (Hb) and globin chain variants, we modified a commonly used method, capillary isoelectric focusing (CIEF), with detection at 280 nm. The samples were hemolysates prepared from red blood cells, and globin chains obtained from the hemolysates by treatment with cold acidified acetone. When the migration time for the internal reference, carbonic anhydrase I (isoelectric point, pI 6.60), was taken as 1.0, the migration ratio for Hb A0 in normal human blood was 0.877 ± 0.004 (mean ± SD, n = 9), and those of the α and β‐ and β‐globin chains were 0.673 ± 0.004 and 0.847 ± 0.005 (mean ± SD, n = 4), respectively. The ratio of peak heights between the β‐ and α‐globin chains (β/α) in the normal Hbs obtained from four subjects was almost constant at 2.5 ± 0.1 (mean ± SD). This ratio indicates which of the globin chains includes a mutation (if one exists). When an Hb variant, Hb Hoshida (in which Gln is substituted for Glu at residue 43 in the β‐globin chain), was analyzed by this method, two main peaks were observed (migration ratios 0.836 and 0.877, corresponding to an abnormal and the normal Hb, respectively). An additional peak with an abnormal migration ratio of 0.788 was also detected in the globin chain profiles. The ratio of peak heights between normal β‐ and α‐globin chains was 1.57, indicating that a mutation exists in the β‐globin chain. We thus established a convenient system using CIEF that provides a rapid and reproducible method for the random analysis of both Hb and globin chain variants.