The author examined the effects of chronic treatment with MK-801 or methamphetamine (MAP) on the muscarinic acetylcholine receptors. Chronic treatment with MK-801 (0, 0.1, 0.5 and 1 mg.kg-1, daily treatment for 7 days) but not MAP produced an increase in total [3H] Quinuclidinyl bensilate (QNB) binding sites (Bmax) in the forebrain in a dose-dependent way without any effect of apparent affinity (the reciprocal of the dissociation constant, Kd). This was associated with the reduction in the behavior sensitivity to scopolamine (1 mg.kg-1, sc) (P < 0.05, at 0.5 and 1 mg.kg-1 compared with the control group). Coadministration of haloperidol (0, 0.125, 0.5 and 2 mg.kg-1), a dopamine receptor antagonist, prevented the up-regulation of muscarinic acetylcholine receptors (mAchR) induced by MK-801 (1 mg.kg-1) in a dose-dependent way, indicating involvement of the dopaminergic mechanism in the induction of up-regulation of mAchRs. The author concludes that chronic treatment with NMDA antagonists but not with MAP produces an up-regulation of mAchRs in the forebrain by facilitating dopamine release and these changes are associated with an alteration in Ach transmission in the central nervous system.

The effects of tumor necrosis factor-alpha (TNF-alpha) on cultured human umbilical vein endothelial cells (EC) and five cancer cell lines, A549, ME180, A2780, KURAMOCHI, and Hela, were compared. While A549, A2780, KURAMOCHI, and Hela cells were fairly resistant to the cytolytic effects of TNF-alpha, ME180 cells were sensitive. EC were also less sensitive to TNF-alpha than ME180 cells as judged by the viability of individual cells and by the release of lactate dehydrogenase (LDH) into the medium. Manganese superoxide dismutase (Mn-SOD) was markedly induced by these cytokines in EC and in A549 cells but not in ME180 cells. The levels of Mn-SOD in the conditioned medium of EC were dramatically increased after stimulation with cytokines, whereas those in ME180 and A549 cells were relatively low. The amount of Mn-SOD released appears to be comparable to that from cells lysed by other means. Immunoblot analysis of Mn-SOD in the medium showed that the molecular mass of the immunoreactive protein was the same as mitochondrial Mn-SOD, indicating that no proteolysis had occurred. These data suggest that in vivo the TNF-alpha produced by cancer cells may induce Mn-SOD in vascular endothelial cells, resulting in release of a relatively large amount of this protein into the serum.

The antimetastatic effect of integrin beta 1 subunit antisense oligonucleotide on human fibrosarcoma cell (HT-1080) in chick embryo was examined. Phosphorothioate modified antisense oligonucleotide (B1-1) was designed to hybridize specifically to integrin beta 1 subunit mRNA. Pretreatment of HT-1080 cells with B1-1 decreased the metastatic ability in chick embryonic liver and lung, while B1-1 showed no toxicity to HT-1080 nor chick embryo. Western blot analysis showed a reduction of the amount of cell surface integrin beta 1 subunit. B1-1 also inhibited HT-1080 attachment to laminin and fibronectin. These results indicate that the inhibition of tumor cell attachment by antisense oligonucleotide might be a good approach to the prevention of cancer metastasis.

The effect of cepharanthin on the enhancement of mitogen-induced histidine decarboxylase (HDC) activity in mice spleens and the effect of histamine on the production of cytokines were investigated. Cepharanthin enhanced LPS-induced HDC activity in normal mice spleens and also enhanced it in genetically T cell-deficient nude mice spleens and T and B cell-deficient scid mice spleens. Therefore, cepharanthin can exert its effect on macrophages without T cells or B cells. Cepharanthin enhanced LPS-induced cytokine production by macrophages. Histamine induced cytokine production and enhanced LPS-induced cytokines production by macrophages. However, diphenhydramine and cimetidine, histamine receptor antagonists, did not block this process. Alpha-fluoromethylhistidine, a suicide inhibitor of HDC, suppressed LPS-induced cytokine production. These results suggest that cytokine production by macrophages is regulated by both histamine added exogenously and histamine induced by macrophages themselves and that histamine participates in enhancement of cytokine production by cepharanthin.

Myometrial contraction induced by oxytocin (OT) is dependent on the OT concentration and the number of OT receptors (OTR) expressed. We previously reported that the number of myometrial OTR expressed is regulated by estradiol and progesterone in myometrial cell culture. In the present study, we evaluate the change in the number of OTR expressed on the cell surface after the addition of OT. After OT was added under various conditions to myometrial cells, we measured the number of OTR expressed on the cell surface (surface-OTR) and those in the plasma membrane (PM) (total-OTR). 1. After the addition of 1 nM OT, total-OTR was 25.78 fmol/mg protein in the OT-negative control and was 23.66 and 7.88 at 30 minutes and 24 hours. 2. Surface-OTR showed no changes after the addition of 1 nM OT at 4 degrees C, and the value after 60 minutes was 8.90 fmol/mg protein. At 37 degrees C, however, it become 5.53 and 1.72 fmol/mg protein 60 minutes after the addition of OT at 10pM and 10nM. This decrease in surface-OTR was suppressed by concanavalin A (ConA), which inhibits the internalization of receptors. Our study demonstrated that OTR expressed on the surface of myometrial cells decreased rapidly in the presence of OT due to internalization.

This study was undertaken to see if estrogen and thyroid hormone affected EGF-receptor (EGF-R) expression, proliferative activity and intracellular SCC levels in uterine cervical squamous carcinoma cells. The uterine cervical cancer cell line (CaSki) was cultured in vitro in the absence or presence of 17 beta-estradiol (E2) or L-triiodothyronine (T3) in a serum free condition. Effects of E2 or T3 on the characteristics of EGF-R were assessed by the Scatchard analysis of the binding assay with 125I-EGF. Cellular levels of EGF-R expression were examined by the immunoperoxidase method with a monoclonal antibody to EGF-R. Proliferative activity of the cells was determined by proliferating cell nuclear antigen (PCNA) immunostaining, 3H-thymidine uptake and the number of cells. The effects of E2 or T3 on intracellular SCC levels were also examined by determining the intracellular SCC concentration with an SCC-RIA Kit. The scatchard analysis of 125I-EGF binding to CaSki cells showed that the addition of E2 or T3 had little effect on the affinity of EGF-R for CaSki cells but increased the capacity of EGF-R for the cells. Immunocytochemical staining with anti EGF-R antibody demonstrated that EGF-R expression in the CaSki cells was augmented by the addition of E2 or T3. The addition of E2 or T3 also resulted in an increase in 3H-thymidine uptake by the CaSki cells, the PCNA positive rate and the number of cells. Furthermore the addition of E2 or T3 increased intracellular SCC in the CaSki cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial lipopolysaccharide (LPS) and other immunostimulants induce an isoform of nitric oxide synthase (iNOS) in vascular smooth muscle (VSM) which produces large quantities of nitric oxide (NO) and profound vasodilation. This process has been implicated as the cause of gram-negative septic shock. It has been demonstrated that interferon-gamma (IFN) markedly potentiates cytokine-induced NO synthesis in various cell types. However, little is known about the mechanism of this enhancing effect of IFN. The present study was undertaken to investigate the effect of IFN on LPS-induced NO synthesis and iNOS mRNA expression in VSM and the possibility of nuclear factor kB (NFkB) involvement in the effect of IFN. LPS-induced NO synthesis is markedly potentiated by IFN in VSM. Expression of iNOS mRNA in VSM costimulated with IFN and LPS was greatly increased compared to that induced by LPS alone. IFN did not alter the lifetime of iNOS mRNA. LPS stimulated translocation into the nuclei of NFkB which is believed to play an important role in the induction of iNOS, but IFN did not enhance NFkB activation by LPS. These results suggest that the enhancing effect of IFN is due to the increased transcription of the iNOS gene rather than a decreased degradation of iNOS mRNA and that the NFkB activation pathway is not involved in this effect of IFN.

IRS-1 (insulin receptor substrate-1) is a major substrate of the insulin receptor. Rat and human IRS-1 cDNAs, and human and mouse IRS-1 genes have been cloned so far. They show high homology in nucleic acids and amino acids levels, which indicate the high conservation of IRS-1 across the species. Interestingly, the entire coding region is contained in the 1st exon in the IRS-1 gene. The promoter of the mouse IRS-1 gene lacks TATA and CAAT boxes but contains 9 potential Spl binding sites, indicating that IRS-1 is a "housekeeping" gene. By deletion analysis, two positively and two negatively regulating fragments are identified in the promoter. In cultured adipocytes, insulin and dexamethasone down regulate IRS-1 expression by different mechanisms. Insulin down regulates at the post-translational level by shortening the protein half life, and dexamethasone down regulates at the post-transcriptional level mainly by shortening the mRNA half life.

Expression of specific genes can be controlled by some compounds (gene expression modifiers) which can be divided into two categories, i.e.; (a) compounds which directly bind to nucleic acids having specific sequences and (b) compounds which bind specific transcription factors (including nuclear receptors) or related factors. Recent results on the structural development and mechanistic studies into the gene expression modifiers, mainly retinoids, phorbol ester, hemin, antisense molecules, and thalidomide analogs, have been reviewed. Some of these gene expression modifiers elicit cell differentiation inducing, tumor promoting, and biological response modifying activities. Development of more potent and/or more specific gene expression modifiers might be useful in cancer chemotherapy.

Cultured human retinoblastoma cells (Y79) were induced to differentiate by dibutyryl cyclic AMP(Bt2cAMP). We examined the effects on the mRNA levels of several cellular genes when the induction of differentiation was monitored by observation of the cellular processes. Bt2cAMP(1mM) treatment produced significant extension of cellular process after 3 days. We examined the mRNA levels of N-myc gene(oncogene), Rb(anti-oncogene, retinoblastoma gene) and nucleolin (nucleolar protein) being linked with ribosome biosynthesis. The mRNA levels of all these genes decreased for 3 days after Bt2cAMP treatment. These results suggested the possibility that Y 79 cells were induced to differentiate by down-modulation of both N-myc gene expression and ribosome biosynthesis in the nucleolus following treatment of Bt2cAMP. Furthermore, the gene expression of the retinoblastoma gene is likely to be downregulated by this condition even if the product of mRNA is not functional.

The accumulation of mesangial matrix is a common histologic abnormality observed over the course of various glomerular disease. To determine the role of growth factors in the mesangial matrix metabolism, the effects of the transforming growth factor-beta (TGF-beta), interleukin-1 beta (IL-1 beta), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), endothelin (ET), and insulin-like growth factor (IGF) on the gene expression of mesangial matrix were studied in cultured rat mesangial cells (MCs). TGF-beta induced transcriptional activation of the genes for alpha 1 (I) and alpha 1 (IV) collagens and fibronectin, and the mRNA levels for these genes in MCs treated with TGF-beta for 24 hours resulted in 1.9-, 2.8-, 2.6-fold increase, respectively, compared to their basal levels. IL-1 beta also transcriptionally increased the mRNA levels for alpha 1 (I) alpha 1 (IV) collagens and fibronectin in the non-stimulated MCs, however, inhibited the increase of these mRNA levels in the TGF-beta-treated MCs. MCs showed little increase in the gene expression for alpha 1 (IV) collagen and fibronectin by the addition of PDGF, EGF, and ET, and no change was noted in the IGF-treated MCs. Detectable gene expression for B1 chain of laminin and heparan-sulphate proteoglycan was observed in the non-stimulated MCs but small in intensity, and no change was noted by stimulation of any growth factors examined.(ABSTRACT TRUNCATED AT 250 WORDS)

The interferon (IFN)-gamma-induced indoleamine 2,3-dioxygenase (IDO) is implicated in the inhibition of intracellular pathogens, e.g. Chlamydia psittaci and Toxoplasma gondii. The intracellular signaling molecules responsible for the induction of IDO gene expression were investigated by the quantitative polymerase chain reaction. The gene expression was inhibited by a tyrosine kinase inhibitor, genistein. Being consistent with this, IFN-gamma induced increased tyrosine phosphorylation and this was inhibited by genistein. The transcription of IDO gene was not inhibited by protein kinase C (PKC) inhibitors, H-7 and staurosporine, or a calmodulin inhibitor, W-7. Irrelevance of PKC in IDO gene expression was supported by the failure of PMA or PMA + A23187 to induce IDO gene expression. These results all suggest that the tyrosine phosphorylation is a critical event in IFN-gamma-inducible IDO gene expression and PKC is not involved in the gene expression.

We examined the effects of cadmium on the mRNA levels of several genes in cultured retinoblastoma (Y79) cells. After Y79 cells were treated with 15 microM CdCl2, RNA was extracted at a given time. The levels of retinoblastoma gene (Rb) mRNA decreased after cadmium treatment, although it was unlikely that the Rb gene product is functional in this cell line. The N-myc gene (oncogene) is constitutively expressed in untreated Y79 cells but its mRNA levels also decreased following cadmium treatment. On the other hand, the mRNA levels of both heat-shock protein (hsp 70) and metallothionein gene, both having physiological protective effects, increased under these conditions. These results indicate that Y79 cells have physiological protective responses to such a heavy metal as cadmium and that both Rb and N-myc gene expressions are down-modulated in the presence of cadmium.