Akira Nakatani

Prion Protein Is Necessary for Latent Learning and Long-Term Memory Retention

Abstract1. The cellular prion protein, designated PrPc, is a key molecule in the prion diseases but its physiological function remains unknown. To elucidate whether PrPc plays some role in the central nervous system, we established a line of mice in which the PrP gene had been disrupted and subsequently conducted long-term observations.2. Performance in latent learning and passive avoidance was evaluated using water-finding and step-through tests, respectively.3. PrP–/– mice showed impaired performance in the water-finding test, indicating a disturbance in latent learning, at 23 weeks of age. In the step-through test, although the PrP–/– mice showed normal learning ability and short-term memory retention, they evidenced a significant disturbance in long-term memory retention.4. These results indicate that PrPc is needed for certain types of learning and memory and that the loss of function of this protein may contribute to the pathogenesis of prion diseases.

Impaired Motor Coordination in Mice Lacking Prion Protein

Abstract1. Prion protein (PrPC) is a host-encoded glycoprotein constitutively expressed on the neuronal cell surface. Accumulation of its protease-resistant isoform is closely related to pathologic changes and prion propagation in the brain tissue of a series of prion diseases. However, the physiological role of PrPC remains to be elucidated.2. After long-term observation, we noted impaired motor coordination and loss of cerebellar Purkinje cells in the aged mice homozygous for a disrupted PrP gene, a finding which strongly suggests that PrPC plays a role in the long-term survival of Purkinje cells.3. We also describe the resistance of the PrP null mice to the prion, indicating the requirement of PrPC for both the development of prion diseases and the prion propagation.

Insulin-like growth factor binding protein measurement: Sodium dodecyl sulfate-stable complexes with insulin-like growth factor in serum prevent accurate assessment of total binding protein content by ligand blotting

The possibility that sodium dodecyl sulfate (SDS)-stable complexes of insulin-like growth factor I (IGF-I) and its binding proteins (IGF-BP) exist in rat serum has been examined by using SDS-polyacrylamide gel electrophoresis (PAGE) followed by both [125I]IGF-I ligand blotting and immunoblotting with antisera directed against either IGF-BP3 or IGF-I. While ligand blotting of rat serum only revealed free IGF-BP subunits (Mr approximately 50, 35, and 30 kDa), immunoblotting with either the IGF-BP3 antiserum or IGF-I antiserum revealed major immunoreactive bands with higher molecular weights (greater than 110, approximately 100, and approximately 84 kDa). The IGF-BP3 antiserum also stained the 50-kDa form of the serum IGF-BP. Specifically stained protein bands were identified by comparison with control immunoblots incubated with normal rabbit serum. Treating the serum with 0.1 N HCl prior to electrophoresis reduced the amount of high molecular weight IGF-BP3 immunoreactive species, with a concomitant increase in the amount of the 50-kDa form. A similar result was obtained if the samples were boiled prior to electrophoresis. These data indicate that not all IGF-BP/IGF complexes may dissociate under normal SDS-PAGE conditions. Therefore, data obtained by using ligand blotting alone may underestimate the amount of total IGF-BP present, especially if the mixture being analyzed also contains large amounts of IGF.

Synaptophysin immunoreactivity in adrenocortical adenomas: a correlation between synaptophysin and CYP17A1 expression

DESIGN AND METHODS The adrenal cortex is not considered to be an intrinsic part of the diffuse neuroendocrine system, but adrenocortical neoplasms possess neuroendocrine properties. In this study, we examined synaptophysin (SYP) and neural cell adhesion molecule (NCAM) expression in adrenocortical adenomas in relation to adrenal function. RESULTS Immunohistochemical analysis showed that 50.7 and 98.6% of the cortical adenomas showed SYP and NCAM immunoreactivities respectively. There was no apparent difference in NCAM immunoreactivity among the adenomas. However, the immunostaining for SYP was significantly stronger in cortisol-producing adenomas (CPA) than in aldosterone-producing adenomas (APA), nonfunctioning adenomas (NFA), showing no clinical or endocrinological abnormality, or adenomas associated with preclinical Cushings syndrome (preCS). Western blotting and real-time PCR demonstrated that the expression level of SYP protein and mRNA was significantly higher in CPA than in APA or NFA. Additionally, the SYP mRNA level showed a positive correlation with CYP17A1 mRNA. In addition to the plasma membrane, mitochondria, and smooth endoplasmic reticulum, SYP immunoreactivity was detected in the Golgi area, which is known to be involved in the regulation of mitochondrial cholesterol and the transport of steroid intermediates. It was unexpected that the ratio of positive cells for SYP in preCS was less than that in APA and NFA. However, further examination is required, because the number of preCS cases we investigated was very small. CONCLUSIONS We propose that SYP expression in adrenocortical cells may be involved in some aspect of adrenal function such as transport or secretion of glucocorticoids.

Tissue specific and cyclic expression of insulin-like growth factor binding proteins-1,-2,-3,-4,-5,-6 in the rat oviduct

Although much is known about the expression insulin-like growth factors (IGF) and their receptors in the murine oviduct, significantly less is known about the expression of IGF binding proteins (IGFBPs). To fill this gap in our knowledge, we identified and characterized the tissue specific expression of IGFBP-1 to-6 in rat oviducts over the estrous cycle byin situ hybridization and immunocytochemistry. Tissues were analysed on proestrus (P1000 h, P2000 h), estrus (E0200, E1000 h), and diestrus I and II (DI 1100 h, DII 1100 h). IGFBP-1 was undetectable in the oviduct over the cycle. IGFBP-2 was selectively expressed in the luminal epithelium. The mRNA levels were high between P2000 h and E1000 h but low or undetectable thereafter. Immunoreactive IGFBP-2 was strong to very strong in these cells over most of the cycle. IGFBP-3 mRNA was undetectable in the oviduct; however, strong hybridization and immunoreactive signals were present in the mesosalpinx and mesotubarium, particularly at DI and DII. IGFBP-4 mRNA was not detected in the oviduct. In contrast, immunoreactive IGFBP-4 was observed in the luminal epithelium and the intensity was very strong after ovulation (E1000 h, DI and DII). IGFBP-5 and-6 mRNAs were selectively expressed in circular smooth muscle cells. Hybridization signals were evident over the cycle, but were greatest at estrus. By comparison, IGFBP-5 and-6 proteins were essentially undetectable in these cells except at DII 1100 h when immunostaining was moderate to high. Luminal epithelial cells were weakly positive for IGFBP-5 and-6. However, intense immunostaining was associated with the ciliated border and the luminal fluid juxtaposed to these cells during the cycle. The oocyte-cumulus complexes were immunostained intensely for IGFBP-2,-4,-5 and-6, but their mRNAs were undetectable. The signals were strongest in degenerating cumulus cells suggesting a potential role for these IGFBPs in cumulus apoptosis. These results demonstrate that the estrous cycle is accompanied by major changes in the pattern of expression of IGFBP-2,-4,-5 and-6 in the rat oviduct. We therefore conclude that the regulated production of these particular IGFBPs may be functionally important in modulating IGF activities in the oviduct, oocyte cumulus complexes, and perhaps the preimplantation embryo as well.

FETAL ASCITES A Report of 3 Autopsy Cases

Three rare autopsy cases of fetal ascites were presented and the etiology of each case was described. Case 1 was a male neonate, delivered by cesarean section at 32 weeks’gestation, and died of respiratory failure. The abdomen was remarkably distended with 1020 ml of ascites. The etiology of Case 1 remained unknown even after macroscopic and microscopic examinations. We considered this as “idiopathic” fetal ascites. Case 2 was a female neonate, delivered at 31 weeks’gestation, with marked abdominal distension and cyanosis. Autopsy revealed 435 ml of ascites, and she was considered to have had “polysplenia syndrome” with cardiovascular malformations. Intrauterine heart failure due to cardiac anomalies was thought to be the cause of this ascites. In case 3 embryotomy was carried out under the diagnosis of fetal ascites by ultrasound examination at 22 weeks’gestation. An urachal cyst connected to the dilated urinary bladder and deficiency of musculature of the abdominal wall composed of loose connective tissue with calcification were observed. The abdominal wall was ruptured and 1,960 ml of ascites was measured. Polycystic kidney with renal dysplasia was also found. Case 3 showed “Prune‐Berry syndrome” and fetal ascites may have arisen from these anomalies.