Immunohistochemical demonstration of DNA polymerase alpha in human brain-tumor cells

The proliferative capacity of brain-tumor cells was analyzed in vitro and in situ using monoclonal antibody (MAb) against deoxyribonucleic acid (DNA) polymerase alpha. For the in vitro studies, two cultured human glioma cell lines were investigated using MAb against DNA polymerase alpha, the MAb Ki-67, a serum against proliferating cell nuclear antigen (PCNA/cyclin), bromodeoxyuridine (BUdR), and an anti-BUdR MAb. During exponential growth of the cells, the percentage of polymerase alpha-positive cells (the "polymerase alpha score") ranged from 72.0% to 77.1%, the Ki-67-positive cells (the "Ki-67 score") ranged from 43.4% to 59.4%, the PCNA/cyclin-positive cells from 30.9% to 41.4%, and the BUdR labeling index from 28.6% to 39.3%. For the in situ studies, tissue from 60 human brain tumors and from two normal human brains was investigated and the polymerase alpha scores and Ki-67 scores were compared. In normal brain tissue, no immunostaining was found by either method. In brain tumors, both the polymerase alpha scores and the Ki-67 scores correlated with the histological grade of malignancy. Polymerase alpha scores were generally higher than Ki-67 scores in the same specimen, especially in malignant brain tumors. These findings suggest that immunostaining of DNA polymerase alpha is a convenient and important new method by which to estimate the cellular proliferation rate of brain tumors. Polymerase alpha scores may be closer to the growth fraction of the individual tumor than the MAb Ki-67 or other scores.     

Oral senile amyloidosis in senescence accelerated mouse (SAM)

Oral senile amyloidosis in senescence accelerated mouse (SAM) was examined for two SAM sublines (P/2/Iw and R/1/Iw) and for various ages by light microscopy, immunohistochemistry, and electron microscopy. The amyloid deposition, identified by green birefrigence following Congo red stain, was observed only in P/2/Iw. In P/2/Iw, no amyloid deposition was found at age 6 months; however, frequency and extent of such deposits increased with advancing age. Distribution of amyloid deposition was as follows: along papillary layers of mucous epithelium in the tongue, the gingiva, the palate, and the buccal mucosa; foci in connective tissues; along vessels, muscles, and minor salivary glands. Immunohistochemically, the amyloid deposition was positive with anti-ASSAM serum being raised against a unique amyloid protein ASSAM which probably induced "senile systemic amyloidosis". P/2/Iw is useful as an animal model of oral senile amyloidosis.     

Elimination of polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) from human blood in the Yusho and Yu-Cheng rice oil poisonings

The pharmacokinetics of polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) in humans was studied by monitoring the blood concentrations of individuals who ingested a contaminated rice oil in Japan (yusho) in 1968 and in Taiwan (yu-cheng) in 1979. Sixteen yusho patients were followed from 1982 to 1990 and three yu-cheng individuals from 1980 to 1989. From the three yucheng patients, blood lipid values for the two persistent toxic congeners, 2,3,4,7,8-pentachlorodibenzofuran (PnCDF) and 1,2,3,4,7,8-hexachlorodibenzofuran (HxCDF), varied from 50 g/kg at first sampling to about 1 g/kg at last sampling corresponding to half-lives for elimination (t_1/2) of 2-2_1/2 years. The blood lipid values for the same PCDF congeners in yusho patients varied from 5 g/kg down to 0.1 g/kg. The calculated t_1/2 were more variable with median values closer to 10 years. Planar PCBs #126 and #169 were present at lower concentrations than the PCDFs. For seven of the other PCB congeners, half-lives for elimination in the yu-cheng individuals varied from 1.2 up to 4.6 yr depending on the degree of chlorination. For the yusho patients, the elimination for the PCBs was longer. These results show that clearance of the toxic PCDFs and PCBs in humans is non-linear with faster elimination at higher exposure followed by slower decreases as background levels are approached. Such a clearance pattern can best be explained by a two compartment liver and fat pharmacokinetic model.     

Accumulation of L-[2-(F-18)]fluorophenylalanine in peri-infarct area in a patient with acute cerebral infarction

We studied the brain uptake of amino acid in a patient with acute cerebral infarction with L-[2-(F-18)] fluorophenylalanine and positron emission tomography. The increased accumulation of the ligand was specifically found in the peri-infarct area where oxygen metabolism was still maintained but decreased later in the 72-day follow-up period. The kinetic analysis revealed that increased accumulation was not due to increased transport from the blood to the brain but to delayed washout from the brain to the blood. Although the mechanism is still unknown, abnormally high accumulation of L-[F-18]fluorophenylalanine may predict delayed neuronal changes after ischemic insults of the brain.     

Poorer nutritional status of the older-generation in a Japanese farming village, as related to fewer types of food consumed

This study was carried out to determine whether the most recent nutritional improvements in Japanese farming villages were due to improvement in the diet of the young only or across all ages. Food duplicates for 24 h were collected. The number of food items and the adequacy of each nutrient level were compared between subgroups, by age of cooking and those eating meals. The older-generation used and ate fewer food items resulting in poorer nutrition. The most recent improvement in nutrition in the farming villages of Japan was found mainly to affect the younger-generation with the older-generations being more poorly nourished.     

Long-term immunological study on postoperative adjuvant treatment with interferon-gamma in patients with renal cell carcinoma who underwent nephrectomy

Previously, we reported the short-term immunological effects of postoperative adjuvant interferon-gamma (IFN-gamma) administration to renal cell carcinoma patients as determined by three-color flow cytometry. We now report the results of a long-term study on a larger number of subjects. Thirty-three patients with renal cell carcinoma received a prophylactic intramuscular injection of IFN-gamma (300 x 10(4) units per week) after nephrectomy. We evaluated immunological changes by measuring peripheral blood lymphocyte subsets including activated cytotoxic T lymphocytes (ACTL), cytotoxic T lymphocytes (CTL), activated suppressor T lymphocytes (ASTL), helper T lymphocytes (HTL), activated suppressor-inducer T lymphocytes (AITL), and suppressor-inducer T lymphocytes (SITL). We also estimated the natural killer (NK) activity by a cytolytic test. All 33 patients were examined for at least 12 months after the start of IFN-gamma injection, and 18 patients were examined for 30 months including the 6-month period following discontinuation of IFN-gamma injection. We found significant enhancement of the ACTL subset from the second week to the sixth month after the start of IFN-gamma injection. On the other hand, we found a significant decrease in the percentage of the HTL and SITL subsets for a long time after the start of injection. NK activity significantly increased throughout the period of administration, and it continued to increase for six months after discontinuation of IFN-gamma injection.     

Lewy body-type degeneration in cardiac plexus in Parkinson's and incidental Lewy body diseases

Heart tissues of patients with PD or incidental Lewy body (LB) disease (ILBD) were examined by light and electron microscopy. LBs and alpha-synuclein-positive neurites were identified in the hearts from 9 of 11 patients with PD and from 7 of 7 patients with ILBD. LBs were present in both tyrosine hydroxylase-positive and -negative nerve processes, which are nerves of extrinsic sympathetic and intrinsic origin, respectively. These findings provide histologic evidence that the postganglionic sympathetic and intrinsic neurons in the heart are involved in the PD disease process.     

Suppression of protein kinase C is associated with inhibition of PYK2 tyrosine phosphorylation and enhancement of PYK2 interaction with Src in thrombin-activated platelets

Blood platelets have recently been shown to express PYK2, a nonreceptor tyrosine kinase belonging to the FAK gene family. In this study, we examined the involvement of protein kinase C (PKC) in PYK2-related responses in human platelets. While PYK2 tyrosine phosphorylation induced by thrombin was inhibited by preincubation of platelets with PKC inhibitors, staurosporine and Ro31-8220, PYK2 association with Src was markedly enhanced under the same conditions. Platelet intracellular Ca²⁺ mobilization induced by thrombin was hardly inhibited by these PKC inhibitors. p130^Cas is a docking protein that associates with FAK or PYK2 through the SH3 domain. Although we identified p130^Cas in platelets for the first time, this docking protein failed to interact with PYK2. These results suggest that PKC activation (but not Ca²⁺ mobilization) is involved in PYK2 tyrosine phosphorylation and that PYK2 associates with Src without PYK2 tyrosine phosphorylation or p130^Cas involvement in platelets.     

Constitutive and cytokine-regulated expression of presenilin-1 and presenilin-2 genes in human neural cell lines

To investigate the role of pleiotropic neuronal and glial cytokines in the regulation of presenilin (PS) gene expression in human neural cells, both presenilin-1 (PS1) and presenilin-2 (PS2) mRNA levels were analysed by Northern blotting in SK-N-SH neuroblastoma, IMR-32 neuroblastoma, NTera2 teratocarcinoma-derived differentiated neurones (NTera2-N) and U-373MG astrocytoma cells following exposure to proinflammatory cytokines (TNF-alpha, IFN-gamma, or IL-1beta), anti-inflammatory cytokines (IL-10 or TGF-beta1), dibutyryl cyclic AMP or phorbol 12-myristate 13-acetate (PMA). The constitutive expression of PS1 (3.0 kb) and PS2 (2.3 kb) mRNA was identified in all these cell lines, in which PS1 mRNA levels were unaltered following treatment with any cytokines and factors examined. By contrast, PS2 mRNA expression was upregulated substantially in SK-N-SH cells by exposure to TNF-alpha and in U-373MG cells by treatment with IFN-gamma, whereas it was downregulated in both NTera2-N and U-373 MG cells following exposure to IL-1beta or PMA. The levels of PS2 mRNA remained unchanged in IMR-32 cells after these treatments. These results indicate that PS1 and PS2 genes are expressed constitutively in a panel of human neural cell lines where PS2 mRNA expression is affected by a distinct set of cytokines via cell type-specific mechanisms that do not alter PS1 mRNA levels, suggesting the existence of separated regulatory systems controlling the expression of PS1 and PS2 genes in human neural cells.