Endogenous tumor necrosis factor exerts its protective function intracellularly against the cytotoxicity of exogenous tumor necrosis factor.

Based on the findings that expression of endogenous tumor necrosis factor (enTNF), which is not present in TNF-susceptible cells, was generally observed in TNF-resistant cells and that TNF gene transfection gives rise to TNF resistance, the assumption was made that enTNF may be a protective protein against the cytotoxicity of exogenous TNF. However, it remains unknown whether the protection by enTNF is exerted in an intracellular or extracellular (autocrine) manner. We therefore transfected a nonsecretory human TNF gene (pTNF delta pro) into highly TNF-sensitive mouse tumorigenic fibroblasts (L-M cells) and investigated their TNF susceptibility. The transfectants expressed enTNF which was not secreted into the medium and acquired an appreciable degree of resistance to exogenous TNF. A significant increase in the manganous superoxide dismutase level was also noted in the transfectants. These findings suggest that enTNF exerts its protective function intracellularly by inducing manganous superoxide dismutase production.     

Development of a new perfusion system for pharmacological study on rabbit basilar arteries

We developed a constant-flow perfusion system to measure vascular responses to vasoactive agents applied intraluminally or extraluminally. The intraluminal and extraluminal sides of a cylindrical section of rabbit basilar artery were isolated completely. By loading with 0.75 g of tension, the resting condition of each preparation was made constant. The intraluminal side was perfused at a constant flow of 8 ml/min and under an intraluminal pressure of 8 mm Hg. When 30 mM KCl was administered intraluminally the preparation showed marked contraction, whereas only slight contraction was observed with extraluminal administration. When 2 x 10(-7) M 5-hydroxytryptamine was administered, no significant differences in contraction could be detected between the intraluminal and extraluminal routes. Application of 10(-6) to 10(-4) M acetylcholine after precontraction with 30 mM KCl resulted in much stronger dilatation upon intraluminal application. Thus, it was demonstrated that under certain conditions significant differences exist in the responses of rabbit basilar arteries to vasoactive agents applied intraluminally or extraluminally. This system can detect the effects of vasoactive agents administered intraluminally and extraluminally at a high level of sensitivity and shows good reproducibility as a means of analyzing vascular functions and characteristics.     

Species and sex differences of aflatoxin B1-induced glutathione S-transferase placental form in single hepatocytes.

Species and sex differences of aflatoxin B1 (AFB1)-induced glutathione S-transferase placental form (GST-P) positive single hepatocytes have been investigated 48 h after an intraperitoneal injection of AFB1 to young male and female Fischer rats (2 mg AFB1/kg body wt) and male Syrian golden hamsters (6 mg AFB1/kg body wt). The presence of GST-P positive hepatocytes was examined by the immunohistochemical method. Male rats formed three times as many AFB1-induced GST-P positive hepatocytes as females. Pretreatment of both male and female rats with an inhibitor of GSH synthesis, buthionine sulfoximine (BSO) (4 mmol/kg body wt), 2 h and 4 h before AFB1 injection increased AFB1-induced GST-P positive hepatocytes by about 120% above the controls. Male hamsters formed several-fold less AFB1-induced GST-P positive hepatocytes than male rats. Pretreatment with BSO did not increase AFB1-induced GST-P positive hepatocytes in hamsters even though it produced an increase in hepatic necrosis. It appears that GSH and GSH S-transferases play an important role in modulating hepatic AFB1-DNA binding and AFB1-induced GST-P positive hepatocytes in rats and hamsters.     

Uptake of 99Tcm-MDP in lung metastasis from osteosarcoma: clinical and animal studies

Bone scintigraphy was performed in 17 patients with previously known lung metastases of osteosarcoma. 99Tcm-MDP uptake was observed in all primary bone lesions but lung metastatic lesions were positive in only six patients (35%). 99Tcm-MDP uptake by lung metastases was significantly correlated with bone and osteoid formation in the metastatic lesions and preoperative serum ALPase values. These clinical observations were confirmed by using nude mice transplanted with human lung metastatic osteosarcoma. 99Tcm-MDP scintigraphy appears to be useful for detecting lung metastases of osteosarcoma only in a selected group of patients.     

Chinese hamster ovary cells produce an enzyme that Nicks heat-labile enterotoxin from enterotoxigenic Escherichia coli

Chinese hamster ovary (CHO) cells were examined for production of an enzyme that nicked the polypeptide chain of the heat-labile enterotoxin from enterotoxigenic Escherichia coli between the A1 and A2 fragments of its A subunit. Serum-free culture medium prepared each day after CHO cell inoculation was concentrated 100 times and its proteolytic activity for formation of the A1 fragment was examined by Western blotting with anti-LT A antibody. The A subunit was detected in culture medium on day 6 after cell inoculation, although not in media on day 1 or 3, indicating that CHO cells produced a nicking enzyme. This nicking enzyme had an optimal pH of about 7.5 and an apparent Mr. of 120,000, as seen by Superose 12 TM gel filtration with an FPLC system. The activity of this enzyme was strongly inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but not by p-chloromercuribenzoic acid, EDTA or ethyleneglycol bis (beta-aminoethylether)-N,N-N',N'-tetraacetic acid, suggesting that this enzyme was a serine protease. The activity was not stimulated by plasminogen or fibrin. These findings suggest that the nicking enzyme was different from proteases such as elastase, collagenase and plasminogen activator, which are probably also secreted by fibroblast-like CHO cells.     

Immunohistochemical localization of Na,K-ATPase in the in situ lens, cultured human lens epithelium and lentoid.

The localization of Na,K-ATPase in the lens is quite controversial. We explored this problem through immunoelectron microscopic examination of rat and human lens. Unlike previously reported results, we have found that Na,K-ATPase is localized in the basal plasma membrane, but not in the lateral or apical plasma membrane of both rat and human lens epithelium. The lens fiber lacked immunoreaction. Localization of Na,K-ATPase was also investigated in the cultured human lens epithelium and in lentoid. Immunoreaction was detected in the apical (facing the media) plasma membrane of the lens epithelium cultured on the lens capsule, whereas the reaction was observable in both apical and basal plasma membrane of the lens epithelium cultured on the biopore membrane filters. Immunoreaction in lentoid was observed in the surface plasma membrane. These data indicate that the polarized distribution seen in the in situ lens epithelium changes when these cells are cultured, and that Na,K-ATPase in the cultured lens cells including lentoid is located in the plasma membrane which is in contact with the growth media. This change in polarity of Na,K-ATPase distribution in cultured epithelial cells may be dictated by the need to maintain ion homeostasis by extrusion of sodium ions across the cell membrane facing the media.     

In vivo measurement of spatial dose distribution with thermoluminescent sheet around high dose-rate intracavitary source: application to rectal cancer.

For intracavitary high dose-rate radiation therapy, a thermoluminescent [TL] sheet for in vivo measurement of spatial dose distribution around source has been recently developed. The TL sheet was found to have a linear response with a very wide dynamic range from at least 0.002 cGy to 5000 cGy for 60Co gamma-rays. This TL sheet (40 cm x 50 cm x 200 microns), which is composed of Teflon mixed with BaSO4:Eu doped powder, is very flexible and can be cut to the desired size. In addition, this sheet is easy to handle because of its insensitivity to room light. The spatial dose distribution is displayed in a color mode by using a newly developed TL sheet readout system. For a clinical application, the TL sheet was wrapped on an applicator for intracavitary radiation therapy of a rectal cancer and was inserted into the rectum. The location of the TL sheet could be confirmed with diagnostic X ray film. After irradiation with high dose-rate 60Co source, the in vivo relative dose distribution on the surface of the rectum was determined. This TL sheet provided a convenient means of measuring the relative dose distributions around 60Co sources of various patterns in intracavitary radiation therapy.