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Parkinson's disease (PD) is an age-related movement disorder that progresses over a period of 10 to 20 years. The existence of microglia in a long-lasting activated state, expressing MHC II, has been thought to play an important role in the progression of PD. PD mouse models, induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), exhibit only transient PD-like movement dysfunction in contrast to MPTP-intoxicated monkeys which show progressive and permanent movement dysfunction. To understand the reasons why the progression does not occur in MPTP-treated mice, we used immunohistochemical analyses to study whether activated microglia in the striatum and/or substantia nigra persist long after MPTP treatment. Microglial changes in the striatum and substantia nigra of mice at 2 days and 6 months after MPTP treatment (four intraperitoneal injections of 20 mg/kg MPTP at two hour intervals) were examined. C57BL/6 mice (which are highly sensitive to MPTP) displayed transient movement dysfunction and highly activated microglia were observed at day two. In contrast, BALB/c mice (which are less sensitive to MPTP) exhibited no movement dysfunction and only slightly activated microglia were observed at day two. After 6 months, the microglia in the striatum and substantia nigra pars compacta of the treated C57BL/6 mice were still more hypertrophic compared with the control, although less hypertrophic than those observed at day two. In the treated BALB/c mice, the microglia were also hypertrophic compared with the control after 6 months. MHC II-positive microglia were undetectable at any time after MPTP treatment in both mice. These data show that MPTP administration results in the existence of persistent activated microglia that are not MHC II-positive, and is independent of the MPTP sensitivity of the mouse strain. These results suggest that long lasting MHC II-positive microglia might be required for PD progression. In MPTP-intoxicated mice, the absence of MHC II-positive microglia might explain why there is no progression of PD-like dysfunctional symptoms.

Glial fibrillary acidic protein (GFAP) was purified from normal bovine brain by a modification of the procedure used to isolate vimentin in order to avoid contamination by other cytoskeletal components: vimentin, neurofilament triplet proteins, tubulin and actin. GFAP is thought to be separated from vimentin in the DE cellulose column chromatography step. The three other major proteins were also separable through ion exchange and gel filtration column chromatographies. A purified 49 kDa polypeptide was estimated to be GFAP from peptide mapping and subsequent immunoblotting analysis. We obtained 4.4 mg GFAP/1 g bovine brain white matter in less than 3 days. The polyclonal antibody raised against purified GFAP was able to detect 49 kDa GFAP by immunoblotting analysis. This isolation method is simpler and more rapid than previous methods.

We have attempted to demonstrate DNA and to measure the amount of nuclear DNA in Pneumocystis carinii by fluorescence microscopy with 4′, 6-diamidino-2-phenylindole (DAPI). Due to the high specificity of DAPI for DNA, only nuclei and mitochondria were stained. An uninucleate trophozoite and a mature cyst containing eight intracystic bodies were distinguished easily each other after staining with DAPI. The total amount of nuclear DNA of eight intracystic bodies in a cyst was approximately eight times that of the small type of trophozoite. When nuclei of uninucleate trophozoites were randomly examined, the distribution pattern of nuclear fluorescence intensity showed two peaks, one at 1C and the other at 2C. Moreover, it showed also a gradual transition in DNA values from 1C to 2C, and very few nuclei with polyploid DNA values. The primary advantage of this method with DAPI is that it allows a more efficient evaluation of the life cycle of P. carinii.

In order to investigate polyploidization of human corneal endothelium, nuclear DNA cytofluorometry was performed. Touch smears of 31 human corneal endothelium taken from 21 autopsies and 10 donor eyes were fixed with absolute methanol and subsequently stained by the pararosaniline-Feulgen nuclear reaction. The optimum condition for Feulgen hydrolysis of the specimens was determined, and all smears were hydrolysed in 1 n HCl for 5 min at 60°C. After the Feulgen reaction, the pararosaniline-DNA fluorescence was measured with an epi-illumination cytofluorometer (Olympus AH-QRFL). Polyploid nuclei, which are the result of abortive cell division after DNA synthesis, appeared with DNA contents of tetraploid (19 cases) and octaploid (two cases) classes. All other nuclei had DNA quantities within the range of the diploid class. Nuclei containing DNA between diploid and tetraploid amounts were not found. It was concluded that most human corneal endothelial cells are of post mitotic G1 population but that a few are in the generative G0 phase.

The suprachiasmatic nucleus (SCN) of mammals is considered to be a circadian oscillator and it also demonstrates circadian rhythmicity of its multiple unit activity. A number of neuropeptides have been found in the SCN. Vasoactive intestinal peptide (VIP)- and vasopressin-containing neurons comprise large populations of these cells and have a distinct distribution within the nucleus. Therefore we attempted to examine whether the VIP neurons show a diurnal alteration of their immunoreactivity by combined immunocytochemistry and color image analysis. Our results demonstrate that VIP-like immunoreactive neurons show a diurnal change in the amount of immunoreactivity. Immunoreactivity was most intense in the sections from rats maintained in the cyclic photoperiod and sacrificed at 02.00 h and weakest in teh SCN from animals sacrificed at 14.00 h. We considered that VIP-like immunoreactive neurons showed diurnal variation of VIP synthesis depending strongly on the light from the retina.

The origin and renewal of the gastrin cells in the pyloric glands of hamsters were studied by [ 3 H]thymidine autoradiography combined with the immunoperoxidase identification technique. After a single injection of [ 3 H]thymidine, the labeling index of gastrin cells was 0.3%, indicating that a few gastrin cells at the isthmus region have a self-replicating activity. Following pulse labeling after five repeated injections of [ 3 H]thymidine at 6-hr intervals, the labeling index of the gastrin cells showed a linear increase (from 1.2% on the 1st day to 5.2% on the 4th day), and attained a plateau (5.6%) on the 5th day. These findings indicate that most gastrin cells differentiate functionally within 5 days. On the 5th day after the last of 41 injections repeated at 6-hr intervals, the labeling index of gastrin cells was 25.8%, and it decreased gradually as follows: 22.3%, 18.3%, 11.2%, 7.3%, 0.9%, and 0% on the 10th, 15th, 20th, 30th, 50th, and 100th day, respectively. From analysis of this decreasing curve, the turnover time of the gastrin cells (a half-life of the cells) was estimated to be 10–15 days. Spatially, the labeled gastrin cells were at first found in a region from the isthmus to the upper level of the glands and later in the lower level of the glands. This indicates that the gastrin cells arising from immature precursor cells at the isthmus region migrate downwards toward the bottom of the glands.

Summary DNA content of diffusely infiltrative carcinomas of 15 Japanese patients, ranging in age from 38 to 65 years, was determined by cytofluorometry, using paraffin sections which were available for histological examination. Sections were stained with 0.0025% propidium iodide. By measuring the fluorescence intensity of at least 50 mitotic cells, ploidy patterns of the cancers were determined, whereas a control of the diploid DNA content was set by measuring the fluorescence intensity of mitotic cells in the non-cancerous gastric mucosa; only metaphase nuclei in the sections were considered to have a whole amount of nuclear DNA in the cytofluorometric measurement. In the present study, it was found that most of the diffusely infiltrative carcinomas consisted of a heteroploid cell line. Their stem DNA contents ranged between 2.8C and 4.8C (2C corresponds to a diploid amount of nuclear DNA). Of 15 cancers, four comprised a mosaic cancer of two different heteroploid cell lines. We encountered only one carcinoma containing a diploid cell line. However, this cancer also contained a heteroploid cell line. Six cancers contained a polyploid cell population, the DNA content of which was double of the stem DNA content. In most cases, the cancer cells distributed in the mucosal layer were also heteroploid, thereby suggesting a heteroploid origin of the diffusely infiltrative carcinomas.

DNA cytofluorometry combined with autoradiography after pulse-labelling with 3 H-TdR visualizes movement of the labelled cells along the cell cycle. If the specimen is fixed after an adequate waiting time, this method enables us to measure the absolute length of the S phase in a cell population with a single sampling. The method was applied for Yoshida sarcoma cells proliferating in the rat ascites. Using a single specimen fixed after 4 h waiting time, the shortest, the average, and the longest durations of S phase in the population were estimated at 5.8, 7.5 and 13 h, respectively. Measurement on a flash-labelled specimen gives the relative durations of G 1, S, G 2 and M. It is shown that, using these 2 samples, a complete cell cycle time analysis can be performed.

Butylated hydroxytoluene (2,6-di-tert-butyl-4-methylphenol, BHT) has been reported to be a lung toxicant. Mice treated with BHT (200-800 mg/kg, po) in combination with an inhibitor of glutathione (GSH) synthesis, buthionine sulfoximine (BOS; 1 hr before and 2 hr after BHT, 4 mmol/kg per dose, ip) developed hepatotoxicity characterized by an increase in serum glutamic pyruvic transaminase (GPT) activity and centrilobular necrosis of hepatocytes. The hepatotoxic response was both time- and dose-dependent. BHT (up to 800 mg/kg) alone produced no evidence of liver injury. As judged by the observation of normal serum GPT, drug metabolism inhibitors such as SKF-525A, piperonyl butoxide, and carbon disulfide prevented the hepatotoxic effect of BHT given in combination with BSO. On the other hand, pretreatment with cedar wood oil resulted in increased hepatic injury in mice treated with both BHT and BSO. Pretreatment with phenobarbital also tended to increase hepatic injury as judged by changes in serum GPT. These results suggest that BHT is activated by a cytochrome-P-450-dependent metabolic reaction and that the hepatotoxic effect is caused by inadequate rates of detoxification of the reactive metabolite in mice depleted of hepatic GSH by BSO administration. The hepatotoxic potencies of BHT-related compounds also were examined in BSO-treated animals. For hepatotoxicity, the phenolic ring must have benzylic hydrogen atoms at the 4 position and an ortho-alkyl group(s) that moderately hinders the hydroxyl group. These structural requirements essentially are the same as those for the toxic potency in the lung (T. Mizutani, I. Ishida, K. Yamamoto, and K. Tajima (1982), 62, 273-281) and support the hypothesis that BHT-quinone methide plays a role in producing liver damage in mice with depressed hepatic GSH levels.