Hybrid cell lines which secreted antibodies to liver-specific membrane lipoprotein (LSP) were obtained by immunizing SMA and BALB/c mice with human LSP and fusing their splenocytes with the myeloma cell line P3-NSI/1-Ag4-1. The secretion of antibody to LSP (anti-LSP) was monitored by binding to a human hepatocellular carcinoma cell line, SK-Hep-1, which possesses surface membrane LSP, and to 125I-antimouse F(ab')2 antibody in radiobinding assay, and by reacting with 125I-LSP in double-antibody radioimmunoassay. From four separate cell fusions, seven secreting hybrids were cloned by dilutional techniques. Of these, four cell lines produced antibodies reacting with a wide variety of cells. The culture supernatants of the remaining three (6D6, 6G3 and 8F10) demonstrated the strongest binding activities against SK-Hep-1 among the various kinds of cell lines tested. However, binding with other cell lines, including renal cancer cells (SK-RC-6) and myeloid cell (HL-60) also occurred. Absorption test of ascitic fluids derived from 6D6 showed that ascitic fluids lost their capacity to bind to target SK-Hep-1 cells when absorbed with SK-Hep-1. Similarly absorption by SK-RC-6 and HL-60 removed almost all of the binding activity of ascitic fluids. Moreover, the binding activities of the ascitic fluids to SK-RC-6 and HL-60 were eliminated when absorbed with SK-RC-6, HL-60 and SK-Hep-1. The present study indicates that our LSP preparation contains nonspecific organ antigens, and although LSP exists on liver cell membrane, it is also found on the cell membrane of other organs albeit in less quantity.

Animals↗

PubMedDate not supplied

Distinct involvement of NF-kappaB and p38 mitogen-activated protein kinase pathways in serum deprivation-mediated stimulation of inducible nitric oxide synthase and its inhibition by 4-hydroxynonenal.

Cytokine-induced expression of inducible nitric oxide synthase (iNOS) and concomitant production of nitric oxide (NO) involve activation of mitogen-activated protein (MAP) kinases and are in most cases mediated by the transcription factor NF-kappaB. We investigated the role of p38 MAP kinase activation and IkappaB phosphorylation in iNOS expression in a novel iNOS-inducing model in mouse macrophages. Deprivation of serum from the culture medium of RAW 264.7 cells up-regulated iNOS and NO production, which were inhibited by 4-hydroxy-2-nonenal (HNE), a component of oxidatively modified low-density lipoprotein (oxLDL). Serum withdrawal induced phosphorylation of Akt, IkappaB, and p38 MAP kinase. Pretreatment with the potent PI3 kinase inhibitor wortmannin, the NF-kappaB inhibitor PDTC or the specific p38 MAP kinase inhibitor SB203580 each partially attenuated the induction of iNOS and NO production, demonstrating that both p38 activation and IkappaB phosphorylation are required for iNOS expression. SB203580, however, did not prevent the phosphorylation of Akt and IkappaB, suggesting that the p38 MAP kinase signal contributes to iNOS gene expression through an IkappaB-phosphorylation-independent pathway. HNE, which markedly inhibited iNOS expression and NO production, prevented the serum withdrawal-triggered IkappaB phosphorylation but not that of Akt or p38 MAP kinase. A high concentration of HNE stimulated dephosphorylation of IkappaB but promoted activation of p38 MAP kinase. Taken together, these results suggest that NF-kappaB and p38 MAP kinase lie in separate signal pathways for serum deprivation-stimulated iNOS expression and NO production. HNE selectively suppresses the former pathway, targeting a site downstream of Akt.

Aldehydes↗

PubMedDate not supplied

Insulin-like growth factor I receptors and insulin-like growth factor-binding proteins in human parathyroid tumors.

Studies have demonstrated the presence of epidermal growth factor receptors in human parathyroid tumors. However, there is little information on the effect of other peptide growth factors on parathyroid cell growth. We therefore studied the interaction of insulin-like growth factor I (IGF-I) with human parathyroid tumor cells. Parathyroid tissues were obtained from 24 patients with primary or secondary hyperparathyroidism. There were 15 solitary adenomas, 5 carcinomas, and 4 hyperplastic tissues. First, the binding of [125I]IGF-I to the crude membrane fractions was studied by competitive inhibition with unlabeled IGF-I. Second, isolated parathyroid cells were cultured with IGF-I and examined for DNA synthesis. The IGF-binding protein (IGFBP) content of tissue homogenates was determined by ligand blot analysis. The binding of [125I]IGF-I to parathyroid membranes was dependent on time, temperature, and pH of the medium. Maximum binding was obtained after incubation for 18 hours at 4 degrees C. Specific binding to parathyroid cancer membranes (mean +/- SE, 10.75 +/- 10.55%/mg protein) was significantly (p < 0.05) greater than that in adenoma tissues (3.71 +/- 2.11%/mg). The value in hyperplastic tissues (4.78 +/- 2.97%/mg) was not different from that in adenomas. Affinity cross-linking and autoradiography demonstrated the type I IGF receptors. Cultured parathyroid cells responded to IGF-I with increased DNA synthesis. The parathyroid tumor tissues expressed IGFBPs. These results suggest that IGF-I and IGFBPs are involved in the growth regulation of parathyroid tumor cells.

Adenoma↗