The effects of sex hormones on the synthesis of prostacyclin (PGI2) by vascular tissues

The effects of estradiol and testosterone on prostacyclin (PGI2) release (measured as 6-keto-PGF1 alpha) by vascular tissues using rat aortic rings and cultured rabbit aortic smooth muscle cells (SMC) were investigated. Aortic SMC were prepared from either explants of atherosclerotic intima or those of normal media. Aortic rings obtained from male and female rats which had been treated with estradiol resulted in increased PGI2 synthesis. Furthermore, PGI2 synthesis by cultured medial SMC was significantly increased in the presence of estradiol (10(-7), 10(-9) M). An increased tendency in PGI2 synthesis was also observed in intimal SMC. On the other hand, aortic rings obtained from female rats treated with testosterone resulted in a significant decrease in PGI2 synthesis. However, aortic rings from testosterone-treated male rats and cultured medial and intimal SMC treated with testosterone (10(-6), 10(-8) M) for 48 hr did not show any significant changes in PGI2 synthesis. We also found greater PGI2 synthesis by intimal SMC compared with that by medial SMC. These results suggest that estradiol and testosterone may have opposite functions in the development of atherosclerosis, that is, estradiol for anti-atherosclerotic and testosterone for atherogenic, by modulating PGI2 synthesis by vascular tissues.     

Change of incidence of antinuclear antibodies in serum of NOD mouse with aging

Extractable nuclear antigens (ENA) were prepared from liver of C57BL/6J mouse and analyzed by SDS PAGE Western-immunoblotting techniques. Some protein components of the ENA, with molecular weights of 94 K, 65 K, 32 K, and 26 K, reacted with antinuclear antibodies in the sera of NOD mice. Incidence of antinuclear antibodies in the sera of NOD mice with aging were measured by ELISA method using the ENA as antigen. The antinuclear antibodies were not detected in young NOD mice (10 weeks old). However, the incidence increased with aging and reached 100% in the female NOD mice of 40 weeks. In the male NOD mice, the incidence of antinuclear antibodies was delayed and low in comparison with that in female.     

Detection of nuclear protein antigens to antinuclear antibodies in serum of NOD mouse

Nuclear protein antigens to the antinuclear antibodies in serum of non-obese diabetic (NOD) mice were investigated. In the serum of diabetic NOD female mice (20 weeks old), the antinuclear antibodies were detected by indirect immunofluorescence assay using frozen sections of liver of C 57 BL/6 J or NOD mice as antigen. Nuclei were separated from the liver of C 57 BL/6 J mice and solubilized. Solubilized nuclear antigens were analyzed by SDS PAGE-Western immunoblotting techniques. Nuclear protein antigens with molecular weights of 26,000, 32,000 and 65,000 showed strongly positive reactions with the antinuclear antibodies in the serum of the NOD mouse.     

Development of nephrotic ICGN mice--the origin, reproductive ability, and incidence of glomerulonephritis

ICGN is a strain of spontaneous nephrotic mice with nonproliferative glomerular lesions. It was derived from an outbred Yok: ICR colony in our laboratory. The renal disease constantly occurred in animals of the first to the tenth generations (greater than 13.0%; 70 days of age). When affected males were mated with unaffected females, the incidence of the disease in their offspring was 38.8% (n = 49) at 70 days after birth. When both parents were affected, their offspring were all affected (n = 12). The disease evenly progressed in both sexes. It usually began 40 to 150 days after birth and death occurred within two months after onset. The animals usually showed sufficient reproductive ability as long as unaffected females were used for mating.     

Isolated ACTH deficiency associated with transient thyrotoxicosis and hyperprolactinemia

A 43-year-old woman with isolated ACTH deficiency in association with transient thyrotoxicosis is reported. The initial evaluation revealed that plasma ACTH and cortisol did not respond to corticotropin-releasing hormone (CRH) in the presence of hyperthyroxinemia and hyperprolactinemia. During the replacement therapy with dexamethasone, she developed transient hypothyroxinemia with persistent hyperprolactinemia. Although thyroid open biopsy did not show any evidence of autoimmune thyroiditis or subacute thyroiditis, the data appear to provide other evidence of a possible relationship between acute adrenal insufficiency and transient thyroid dysfunction.     

Identification and partial purification of a novel tumor-derived protein that induces tissue factor on cultured human endothelial cells

Conditioned medium of a human bladder carcinoma cell line (J82) was found to induce tissue factor synthesis in cultured human umbilical vein endothelial cells (HUVEC). A protein present in the J82 conditioned medium was partially purified by FPLC using a combination of MONO Q and Superose 6 columns. The bladder carcinoma-derived cytokine (BCDC) exhibited a Mr of 22 kDa by gel permeation HPLC. Polyclonal antibody against either interleukin-1, tumor necrosis factor, or transforming growth factor-beta failed to inhibit the ability of the conditioned medium to induce HUVEC tissue factor activity, suggesting that this tumor cell line secretes a novel cytokine responsible for HUVEC tissue factor induction.     

Expression of active alpha-3 subunit of rat brain Na+,K+-ATPase from the messenger RNA injected into Xenopus oocytes

Cloned cDNA encoding the so-far uncharacterized alpha-3 subunit of rat brain Na+,K+-ATPase (Hara et al. (1987) J. Biochem. 102, 43-58, Shull et al. (1986) Biochemistry 25, 8125-8132) was incorporated into a vector carrying the SP6 promoter. The mRNA produced in vitro was injected into Xenopus oocytes with the mRNA encoding the Na+,K+-ATPase beta subunit of Torpedo electroplax. Increased Na+,K+-ATPase activity in the oocyte membrane was observed. This newly expressed activity was inhibited by ouabain (Ki = 1.5 x 10(-7) M), suggesting that the alpha-3 subunit of rat brain Na+,K+-ATPase is a highly ouabain-sensitive catalytic subunit.     

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study

1H NMR spectra of a GDP/GTP-binding domain of human c-Ha-ras gene product (residues 1-171) in which glutamine-61 was replaced by leucine [ras(L61/1-171) protein] were analyzed. By one-dimensional and two-dimensional homonuclear Hartmann-Hahn spectroscopy and nuclear Overhauser effect (NOE) spectroscopy of the complex of the ras(L61/1-171) protein and GDP, the ribose H1', H2', H3', and H4' proton resonances of the bound GDP were identified. The guanine H8 proton resonance of the bound GDP was identified by substituting [8-2H]GDP for GDP. The dependences of the H1' and H8 proton resonance intensities on the duration of irradiation of the H1', H2', H3', and H8 protons were measured. By numerical simulation of these time-dependent NOE profiles, the conformation of the protein-bound GDP was elucidated; the guanosine moiety takes the anti form about the N-glycosidic bond with a dihedral angle of chi = -124 +/- 2 degrees and the ribose ring takes the C2'-endo form. Such an analysis of the conformation of a guanine nucleotide as bound to a GTP-binding protein will be useful for further studies on the molecular mechanism of the conformational activation of ras proteins on ligand substitution of GDP with GTP.     

Lysing of fresh human tumor by a cytotoxic factor derived from autologous large granular lymphocytes independently of other known cytokines

Summary During interaction with autologous tumor cells large granular lymphocytes (LGL) of cancer patients released a soluble cytotoxic factor, termed LGL-derived cytotoxic factor, which mediated lysing of autologous fresh tumor cells. The cytotoxic factor was compared with purified human recombinant cytotoxic cytokines, including tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN) , IFN, interleukin-1 (IL-1) and IL-2. The LGL cytotoxic factor exhibited cytotoxicity against autologous and allogeneic fresh human tumor cells in an 18-h⁵¹Cr-release assay, while these target cells were resistant to lysing by any of the recombinant cytokines. Mixtures of recombinant(r) TNF, rLT, rIFN, rIFN, rIL-1 and rIL-2 were still unable to produce cytotoxic effects on fresh human tumor cells. Treatment with monoclonal and polyclonal antibodies directed against rTNF, rLT, rIFN, rIFN, or rIL-1 did not inhibit the cytotoxic activity of LGL-derived cytotoxic factor against fresh human tumor cells. Even a mixture of all the antibodies was incapable of blocking the cytolytic activity of the factor to fresh human tumor cells. Furthermore, intact LGL-mediated lysing of autologous tumor cells was not inhibited by any of the antibodies. These results may indicate that a cytotoxic factor produced by LGL in response to autologous tumor cells mediates lysing of fresh human tumor cells independently of TNF, LT, IFN, IL-1 and IL-2.     

Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.