Haruo Shinohara

αB crystallin and HSP28 are enhanced in the cerebral cortex of patients with Alzheimer's disease

Abstract The localization of two small heat shock proteins, αB crystallin and 28-kDa heat shock protein (HSP28), was studied immunochemically and immunohistochemically in cerebral cortex of patients with Alzheimers disease (AD) and control groups. By specific immunoassays for αB crystallin and HSP28, we found that the concentrations of αB crystallin are elevated in the temporal and frontal lobes, while those of HSP28 are elevated in the temporal, frontal, and parietal lobes in the AD brains. Immunohistochemically, αB crystallin was localized in astrocytes and oligodendrocytes, while HSP28 was present in degenerating neurons in the cerebral cortex of both AD and control brains. However, αB crystallin-immunoreactive astrocytes and HSP28-immunoreactive degenerating neurons were more frequently observed in AD brains. The immunoreactivity for HSP28 was also found in the senile plaques in the AD brains. These findings suggest that the increased accumulations of these small heat shock proteins appear to be part of reactive processes of glial cells and neurons under pathologic conditions.

Developmental and Age-Dependent Changes of 28-kDa Calbindin-D in the Central Nervous Tissue Determined with a Sensitive Immunoassay Method

Abstract: For the quantitative analysis of vitamin D‐dependent 28‐kDa calcium‐binding protein (calbindin‐D) in the CNS, we have established a highly sensitive immunoassay method. The antisera were raised in rabbits with purified calbindin‐D from rat kidneys, and the antibodies were purified with a calbindin‐D‐coupled Sepharose column. The purified antibodies were specific for calbindin‐D, showing a single band on the immunoblot with the extract of rat kidney or cerebellum. The sandwich‐type immunoassay system was prepared by the use of purified monospecific antibodies, and the minimum detection limit of the assay was 0.1 pg or 3.6 amol of calbindin‐D, which was sufficiently sensitive for the measurement of calbindin‐D content in isolated Purkinje cell bodies at the level of single cells. The average content of calbindin‐D in a single Purkinje cell was 0.05 pg. Calbindin‐D was detected in most of the rat tissues examined, but it was present predominantly in the kidney and CNS, especially in the cerebellum. Calbindin‐D was detected at a similarly low level in the cerebral cortex, cerebellum, and brainstem of rat embryos of 15 gestational days, and it increased gradually but differently in these regions, reaching the respective adult levels by 4–5 weeks of postnatal age. In contrast, kidney calbindin‐D increased sharply between 15 gestational days and 3 postnatal days, reaching the adult level by 6 days of age. Calbindin‐D levels in the adult rat CNS were affected little by age, whereas the concentrations in human cerebral cortices were significantly low in the aged brain as compared with those in the young brain. However, the concentrations in various regions of cerebral cortex from patients with Alzheimers disease showed values similar to those in the relevant regions of the age‐matched control patients.

Translocation and induction of αB crystallin by heat shock in rat glioma (GA-1) cells

Abstract Response to heat shock of αB crystallin expressed in rat astrocytoma GA-1 cells was analysed quantitatively using an immunoassay method. GA-1 cells contained a considerable amount of αB crystallin at growing phase. When the growing cells were heated at 45°C, concentrations of αB crystallin in cell extracts were decreased to less than one-fifth of the original level within 15 min, with an increase in the insoluble fraction which was detected by immunoblotting. The low level of αB crystallin in the cytoplasm, that was observed for a few hours after heat shock, gradually recovered to the control level within several hours. At 10 h following heat shock (45°C for 15 min), the concentration of αB crystallin in the soluble extract was about twice that of the control level, with little detectable amounts in the insoluble fraction. These results are additional evidence that suggest that αB crystallin is a small heat shock protein.

High Expression of the γ5 Isoform of G Protein in Neuroepithelial Cells and Its Replacement of the γ2 Isoform During Neuronal Differentiation in the Rat Brain

Abstract : High concentrations of G proteins, which include multiple isoforms of each subunit, α, β, and γ, are expressed in the adult brain. In this study, we concentrated attention on changes of these isoforms during embryonic development in the rat brain. Concentrations of γ2 as well as GoAα, GoBα, and β2 were low in early embryogenesis and then increased, whereas expression of γ5, in contrast, was initially high followed by a drop, with only very low levels observed throughout postnatal development. Among the other isoforms, Gi1α, Gsα‐short, G12α, G13α, β4, γ3, γ7, and γ12 were present in the embryonic brain at low levels, but their levels markedly increased after birth. In contrast, the levels of Gi2α, Gsα‐long, Gq/11α, and β1 were essentially constant throughout. Immunohistochemical staining of the brain vesicles in the embryos showed γ5 to be specifically expressed in the proliferative region of the ventricular zone, whereas γ2 was mainly present in differentiated neuronal cells of the marginal zone. Furthermore, differentiation of P19 mouse embryonal carcinoma cells to neuronal cells with retinoic acid induced the expression of γ2 and a decrease of γ5, the major isoform in the undifferentiated state. These results suggest that neuronal differentiation is responsible for the on/off switch of the expression of γ2 and γ5 subunits.

Differential localization of G proteins, Gi and Go, in the olfactory epithelium and the main olfactory bulb of the rat

The immunohistochemical localization of proteins Gi1 (plus Gi3). Gi2 and Go was studied in the olfactory epithelium and the main olfactory bulb of rats, using purified antibodies to the respective alpha subunits and beta gamma subunits of these G proteins. In the olfactory epithelium, only a restricted population of olfactory cells was immunopositive for Gi2 alpha, but others were not. The immunoreactivity for Gi1 alpha/Gi3 alpha was not observed. The olfactory epithelium was immunopositive for both Go alpha and beta gamma, but its apical surface was immunopositive only for beta gamma. In the main olfactory bulb, all layers were intensely immunopositive for Go alpha and beta gamma but weakly for Gi2 alpha. In contrast to the negative or weak immunostainings in the olfactory nerve fiber layer and glomeruli, the molecular and the internal granular layers were intensely immunopositive for Gi1 alpha/Gi3 alpha. These findings suggest the functional difference among Gi1/Gi3, Gi2 and Go in the signal transduction in the olfactory system.

Immunoreactive parvalbumin concentrations in parahippocampal gyrus decrease in patients with alzheimer's disease

By using a sensitive enzyme immunoassay system for rat parvalbumin, we determined parvalbumin contents in the 4 cerebrocortical regions (superior frontal gyrus of frontal lobe, parahippocampal gyrus of temporal lobe, superior parietal lobule of parietal lobe, and calcarine area of occipital lobe) of patients with Alzheimers disease and age-matched controls. Among the 4 regions, concentrations of parvalbumin were the highest in calcarine area (68.6 +/- 6.7 ng/mg protein, rat parvalbumin equivalents, mean +/- SE) and the lowest in the parahippocampal gyrus (11.0 +/- 1.7 ng/mg protein) in the controls. A similar regional difference of the concentration was observed also in the patients with Alzheimers disease. When compared with the controls, however, concentrations of parvalbumin in parahippocampal gyrus of patients with Alzheimers disease (4.0 +/- 0.9 ng/mg protein) were significantly low (P less than 0.01), showing less than a half of the control values. In contrast, the concentrations in the 3 other regions showed little difference between Alzheimers disease and the controls.

Development of the Innervation Pattern in the Upper Limb of Staged Human Embryos

The upper limb nerves of 8 human embryos (Carnegie stages 13-21) were studied by reconstruction. In stage 13, upper limb nerves (C5-T1) extended from the spinal cord. In stage 14, these nerves united to form the nascent brachial plexus. In stages 16 and 17, the median nerve, the radial nerve and the ulnar nerve entered into the hand plate. In stages 20 and 21, the upper limb nerves were observed in an orientation and arrangement similar to those in the adult.

Responses to heat shock of αB crystallin and HSP28 in U373 MG human glioma cells

Abstract Responses to heat shock of αB crystallin and small heat-shock protein HSP28, which are expressed at considerable levels in human astroglioma U373 MG cells (2–4 μg of each per mg soluble protein in confluent cultures), were analysed quantitatively by specific immunoassays. Concentrations of αB crystallin and HSP28 in soluble extracts of U373 MG cells decreased to about 50% of original values, with an increase in the insoluble fraction, during heat treatment for 15 min at 45°C. The concentrations of αB crystallin and HSP28 increased gradually upon return to 37°C, reaching and then exceeding the control levels within 5 h and 10 h, respectively, after heat shock. During centrifugation on sucrose density gradients both αB crystallin and HSP28 in extracts from untreated and heat-treated cells sedimented at the same position, which corresponded to a molecular mass of > 540 kDa . This result suggests that the sizes of aggregates of the two proteins in the cytoplasm are not affected by heat shock. Both αB crystallin and HSP28 in an extract of U373 MG cells were trapped on and coeluted from an affinity column prepared with antibodies against αB crystallin. These results suggest that the two proteins are also associated in U373 MG cells.

Sensitive immunoassay for rat parvalbumin: tissue distribution and developmental changes

A sensitive enzyme immunoassay for measurements of rat parvalbumin was established using antibodies raised in rabbits with parvalbumin purified from skeletal muscles. Antibodies in the antiserum were purified with a parvalbumin-coupled Sepharose column. The sandwich-type immunoassay system for parvalbumin was composed of polystyrene balls with immobilized purified antibodies and the same antibodies labeled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive and the minimum detection limit was 1 pg parvalbumin/tube. The assay did not cross-react with other calcium binding proteins, including human S-100a0 and S-100b proteins, rat 28-kDa calbindin-D, and bovine calmodulin. High concentrations of parvalbumin were observed in the skeletal muscles, especially in those composed of fast-twitch fibers, and in the diaphragm and tongue, but not in heart muscle. A relatively high concentration was estimated in the central nervous tissue. Parvalbumin was detected in the cerebral cortex and cerebellum of gestational 15-day fetuses. However, the levels of parvalbumin in the muscle tissues and central nervous tissue were very low in rats before 1 week of age. Thereafter, they increased sharply, reaching the adult levels by 5 weeks in most of the tissues. Parvalbumin concentrations in adult rat soleus muscle increased less than 20-fold within 10 days after transection of the ipsilateral sciatic nerve, while the concentrations in the extensor digitorum longus muscle did not change in the same period.

Localization of a G protein Gi2 in the cilia of rat ependyma, oviduct and trachea

In previous studies, the localization of a pertussis toxin‐sensitive G protein was demonstrated in ependymal cilia, but the identification of the subtype of G protein was inconsistent. To clarify this issue, we studied the localization of Goα, Gi1α, Gi3α and Gi2α in the ciliated ependymal cells and in the cilia of some other tissues of rats using specific antibodies. The cilia of the ependymal cells that line the ventricular cavity of the brain were intensely immunoreactive for Gi2α, but not for Goα, Gi1α or Gi3α. Immunoblot analysis demonstrated higher levels of Gi2α in the ependymal cilia‐rich pellet than in the motor area of the parietal cortex. At the ultrastructural level, the immunoreactivity specific for Gi2α was found predominantly in the cilia, but rarely in the microvilli or the basal bodies of ependymal cells. In cross‐sections, the immunoreactivity specific for Gi2α was observed only in cell membranes, in particular, in the inner electron‐dense leaflet of the trilaminar structure. In addition to that in the ependymal cilia, such specific localization of Gi2α was observed in the motile cilia in other tissues, including the oviduct and trachea. By contrast, the stereocilia in the ductus deferens were not immunopositive for Gi2α. These findings suggest that Gi2 might play an important role in the signal transduction in ciliary membrane‐associated function(s) of the ependymal cells, oviduct and trachea.