Differential expression of the beta I- and beta II-PKC subspecies in the postnatal developing rat brain; an immunocytochemical study.

Differential expression of protein kinase C subspecies, beta I- and beta II-PKC, derived from a single gene by alternative splicing was evidenced in the postnatal developing rat brain. Immunoblot analysis of the PKC subspecies in the whole developing brain showed that beta I-PKC was present at birth and then gradually increased, while beta II-PKC was not present at birth or on postnatal day 3, then increased rapidly from day 7 to the maximum value seen in the adult brain. Under light microscopy, beta I-PKC immunoreactivities seen at birth were the most intense in the brainstem and intense in the diagonal bundle and globus pallidus. beta I-PKC immunoreactivities in these neurons weakened from day 7 and disappeared in the adult brain, while in the cerebral cortex, triangular septal nucleus and pontine nucleus beta I-PKC immunoreactivities were week at birth and then gradually increased. beta II-PKC immunoreactivities were first visible in neurons on day 7 and increased progressively. beta I- and beta II-PKCs were not co-localized in a neuron, as far as examined. The immunoreactivities of beta I-PKC at birth were localized in growth cone-like structures as well as in the dendrites and perikarya. Similarly, alpha-PKC was also present at birth in the growth cone-like structure. Immunoblot analysis revealed that beta I-PKC was present at birth in the growth cone-rich fraction from the hindbrain but not in that from the forebrain, while alpha-PKC was found in the growth cone-rich fraction from both the forebrain and the hindbrain. beta II- and gamma-PKC were not detected in the growth cone-rich fraction from either forebrain or hindbrain. These findings suggest that beta I- and beta II-PKC play a role in different stages of development and in different neurons; both beta-subspecies may be involved in postnatal developing neuronal functions while only beta I-PKC plays functional roles in the growth cone, in the prenatal developmental stage.     

Immunohistological study on the expression of proliferating cell nuclear antigen (PCNA/cyclin) in human colorectal lesions]

The purpose of this study was to clarify the significance of immunohistological staining for PCNA/cyclin in human colorectal lesions. Our results: The PCNA-positive cells existed at the bottom of colonic tubuli in the normal and hyperplastic conditions. In the neoplastic lesions, however, the positive cells were existed at the relatively surface of the mucosa (chi 2: P less than 0.01) and distributed irregularly from the bottom to the top of carcinoma tissue. These results suggested that immunohistological staining for PCNA would specifically detect the cell proliferation and be beneficial for practical use and clinical application of the diagnosis of the colorectal lesions.     

Purification of rabbit, rat and mouse protein C with the use of monoclonal antibody to human protein C, PC01

Ca(++)-dependent monoclonal antibody specific to gamma-carboxyglutamic acid (Gla) domain of protein C was produced. It did not cross-react to the other vitamin K-dependent plasma proteins but to protein C of the other species. Using this monoclonal antibody, PC01, rabbit (170 micrograms), rat (60 micrograms) and mouse (40 micrograms) protein Cs were isolated from 100 ml of their plasma by affinity chromatography. All of these protein Cs were two chain form linked by disulfide bond as well as human protein C and activated by thrombin-thrombomodulin complex. Rat and mouse protein Cs showed similar characters to human protein C. On the other hand rabbit protein C had different M(r) of heavy and light chains and showed lower anticoagulant activity compared with human protein C.     

Phosphorylation of neurofilament H subunit as related to arrangement of neurofilaments

To find out what causes differences in phosphorylation states in neurofilaments (NF), we selected two types of dendrite, one provided with very few NFs (Purkinje cell) and the other with relatively many (anterior horn cell). We examined these with four monoclonal antibodies selected by the Western blot analysis, two (NE14 and SMI31) recongnizing only phosphorylated, SMI32 recognizing only nonphosphorylated, and N52 recognizing phosphorylation-independent epitopes of NF-H. The immunoperoxidase labeling of dendrites, and also of perikarya, in both neurons was detectable with all four antibodies. After the tissue was treated with Triton X-100, the labeling was still detectable with SMI32 or N52, but undetectable with NE14 and SMI31. The brain homogenate Triton-extracted supernatant after centrifugation at 100,000g for 1 hr showed the staining of NE14, SMI31, and N52 but not that of SMI32. In Purkinje cell dendrite and perikaryon, NFs always appeared singly. In the immunogold labeling, they were labeled only with SMI32 or N52. Labeling by NE14 or SMI31 was distributed throughout the cytoplasm and hardly associated with NFs. In the anterior horn cell dendrite and perikaryon, NFs appeared both single and in bundles. They were predominantly labeled with SMI31 or N52 when they were single, and with NE14, SMI31, or N52 when they were bundled. Even in one NF, portions that appeared single were labeled mostly with SMI32 or N52, while the remainder, to which other NFs approached closely, were labeled mostly with NE14, SMI31, or N52. Thus, when NFs appear singly, NF-H in their projections or cross-bridges with other organelles is not phosphorylated, while when NFs are bundled, NF-H is phosphorylated in crossbridges between NF core filaments. These data may explain why the NF-H is heavily phosphorylated in axons, where NFs are abundant, and not in dendrites and perikarya, where NFs are sparse. Wiley-Liss, Inc.     

Polyclonal and monoclonal antibodies monospecific to MMTV LTR orf protein produced in E. coli.

Monoclonal and polyclonal antibodies specific to an open reading frame of the mouse mammary tumor virus long terminal repeat were generated using an open reading frame-beta-galactosidase fusion protein produced in E. coli. Both antibodies reacted with the open reading frame-beta-galactosidase fusion protein but not with beta-galactosidase alone using an immunoblotting technique. It is concluded that these antibodies were specific for the protein encoded by the open reading frame of the mouse mammary tumor virus long terminal repeat.     

I.v. fentanyl decreases the clearance of midazolam

We have examined the effect of fentanyl on the pharmacokinetics of midazolam in patients undergoing orthopaedic surgery. Thirty patients were allocated randomly to receive fentanyl 200 micrograms and midazolam 0.2 mg kg-1 (fentanyl group, n = 15) or placebo and midazolam 0.2 mg kg-1 (placebo group, n = 15) in a double-blind manner for induction of anaesthesia. Anaesthesia was maintained with nitrous oxide and isoflurane. Systemic clearance of midazolam was decreased by 30% (P = 0.002) and elimination half-time was prolonged by 50% (P = 0.04) in the fentanyl group compared with the placebo group. There were no differences in the distribution half-time or volume of distribution at steady state between the two groups. These findings indicate that elimination of midazolam was inhibited by fentanyl during general anaesthesia.      

Analysis of long-term social rehabilitation of brain contusion]

164 patients with brain contusion were evaluated with respect to social rehabilitation. 70 out of 134 patients (66.7%), said to have had good recovery or moderate disability by the Glasgow Outcome Scale (GOS), returned to full or partial employment. Factors such as age, Glasgow Coma Scale (GCS) at admission, duration of unawareness, fibrin and fibrinogen degradation product (FDP) were the most important in predicting social recovery. The Wechsler Adult Intelligence Scale (WAIS) was applied in 33 patients. The IQs of the patients who returned to their job fully tended to be higher than those who could not. In the majority of patients, impaired capacity for work was caused not only by physical deficits, but by mental retardation described as such as IQ score.     

Suppressive effect of vitamin E on lipid peroxidation in halothane-administered guinea pig liver.

The effect of vitamin E on halothane-induced liver damage was studied in guinea pig halothane hepatitis. Twenty animals were divided into 3 groups, consisting of a control group, a halothane group and a vitamin E + halothane (H) group. The animals in the control group (n = 6) were allowed to inhale air only. The animals in the halothane group (n = 6) and the vitamin E + H group (n = 8) were allowed to inhale 1% halothane with air. Animals in the vitamin E + H group were additionally injected with 30 mg kg-1 of vitamin E 30 minutes prior to inhalation of halothane. Blood was aspirated from the heart immediately after sacrificing to measure the serum activity of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT). A microsomal suspension was prepared from the excised liver. Then the amount of thiobarbituric acid (TBA) reactive products in the microsomes were measured. The amount of tissue TBA-reactive products was increased by inhalation of halothane. The increase in the amount of TBA-reactive product was inhibited by the administration of vitamin E. The serum GPT activity was increased by halothane inhalation. Increased serum GOT and GPT activity were inhibited by the administration of vitamin E. These results demonstrated that vitamin E suppressed halothane-induced liver damage in the guinea pig by inhibiting lipid peroxidation.     

Presence of ghost cells and the Wnt signaling pathway in odontomas

BACKGROUND: Although it has been reported that ghost cells are present in odontomas, the generation mechanism of these cells is unclear. To evaluate the presence of ghost cells and involvement of the Wnt signaling pathway, we examined the expression of hard keratins, beta-catenin and Lef-1 in odontomas. METHODS: Sixty-nine cases of odontoma were examined immunohistochemically with the use of antibodies against human hair proteins, beta-catenin and Lef-1. RESULTS: Expression of hard keratins was found only in the cytoplasm of ghost cells in 46 (66.7%) of the 69 odontomas. Compound odontomas (78.8%) showed a higher incidence of ghost cells than complex odontomas (29.4%). Histopathologically, ghost cells were found within odontogenic epithelium adjacent to immature enamel and in the centre of Liesegang-ring-like calcified materials. Expression of beta-catenin and Lef-1 was observed in the cytoplasm and nucleus of odontogenic epithelial cells adjacent to the ghost cells in immature odontomas. CONCLUSION: These findings suggest that odontoma is a hard keratin-expressing tumor-like lesion, and that the Wnt signaling pathway may be involved in the formation of ghost cells in odontomas.