Presence of a genistein-responsive inhibitory mechanism on interleukin-1alpha-induced NF-kappaB activation.

Interleukin 1 (IL-1) is known to activate the signal transduction machinery, including the transcription factor, nuclear factor kappa B (NF-kappaB). The activation mechanism of NF-kappaB has been studied intensively, while the negative regulatory mechanisms of NF-kappaB remain to be clarified. In the present study, we found that genistein, a tyrosine kinase inhibitor, augmented IL-1alpha-dependent NF-kappaB activation, suggesting the presence of a tyrosine kinase mediating a suppression signal on NF-kappaB. As determined by luciferase reporter gene assay using kappaB-responsive element, genistein enhanced IL-1alpha-induced NF-kappaB activation. Although genistein failed to increase luciferase activity at 1 and 3 h after IL-1alpha stimulation, it induced prolonged activation beginning at 6 h after the initial stimulation. We next examined whether genistein augmented the DNA-binding activity of NF-kappaB, using electrophoretic mobility shift assay. In the case of the control experiment, the binding of NF- kappaB to the kappaB-responsive element peaked at 30 min after IL-1alpha stimulation, and decreased thereafter. In contrast, treatment with genistein maintained the maximum binding activity for at least 2 h after stimulation. Moreover, genistein enhanced the IL-1alpha-dependent degradation of IkappaBalpha. Taken together, our results indicate that genistein augments IkappaB degradation, resulting in continuous NF-kappaB activation. This suggests the possibility that tyrosine kinase negatively regulates NF-kappaB.

Differential involvement of p38 mitogen-activated protein kinase and phosphatidyl inositol 3-kinase in the IL-1-mediated NF-kappa B and AP-1 activation.

Interleukin-1 (IL-1) is a central regulator of the immune and inflammatory responses by which various inflammatory genes are induced. Although IL-1 signaling is known to involve PI3-kinase, p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK), the crosstalk of these kinases on the IL-1-mediated signal transduction is not clear. We used two specific inhibitors, SB203580 which selectively inhibits p38 MAP kinase and LY294002 which inhibits PI3-kinase, respectively, to explore the involvement of these kinases in the IL-1-induced NF-kappa B activation, using a human glioblastoma cell line, T98G. Two kinase inhibitors decreased IL-1-induced IL-8 mRNA and protein levels markedly. IL-1 caused phosphorylation of p38 MAP kinase with concomitant recruitment of PI3-kinase to IL-1 receptor I (IL-1RI) and its activation. In this context, pretreatment of LY294002, but not SB203580, inhibited IL-1-induced NF-kappa B activation significantly. While IL-1 induced-AP-1 activation was moderate, both LY294002 and SB203580 suppressed IL-1-induced AP-1 activation. These observations were prominent particularly in the TRAF6 transfection system, in which overexpression of wild type TRAF6 augmented the IL-1 mediated NF-kappa B and AP-1 activation, while dominant negative TRAF6 construct (delta TRAF6) suppressed these activation. Namely, LY294002 inhibited TRAF6-mediated IL-1-induced NF-kappa B and AP-1 activation markedly, while SB203580 inhibited TRAF6-induced AP-1 activation but not NF-kappa B activation. Above results indicated that both PI3-kinase and p38 MAP kinase are differentially involved in IL-1-induced NF-kappa B and AP-1 activation.

A MEK inhibitor, PD98059 enhances IL-1-induced NF-kappaB activation by the enhanced and sustained degradation of IkappaBalpha.

Interleukin-1 (IL-1) mediates numerous host responses through rapid activation of nuclear factor-kappaB (NF-kappaB), but signal pathways leading to the NF-kappaB activation appear to be complicated and multiplex. We propose a novel regulatory system for NF-kappaB activation by the extracellular signal-related kinase (ERK) pathway. In a human glioblastoma cell line, T98G, IL-1-induced NF-kappaB activation was significantly augmented by the pretreatment of a specific MEK inhibitor, PD98059. In contrast, ectopic expression of a constitutive activated form of Raf (v-Raf) reduced IL-1-induced NF-kappaB activation, and this inhibition was completely reversed by PD98059. Interestingly, PD98059 sustained IL-1-induced NF-kappaB DNA binding activity by an electrophoretic mobility shift assay and also IkappaBalpha degradation, presumably by augmenting and sustaining the proteasome activation. Concomitantly, two NF-kappaB dependent genes, A20 and IkappaBalpha expression were prolonged with PD98059. These data suggested that MEK-ERK pathway exerts a regulatory effect on NF-kappaB activation, providing a novel insight on the role of MEK-ERK pathway.

The N-terminal helix of Xenopus cyclins A and B contributes to binding specificity of the cyclin-CDK complex.

Mitotic cyclins A and B contain a conserved N-terminal helix upstream of the cyclin box fold that contributes to a significant interface between cyclin and cyclin-dependent kinase (CDK). To address its contribution on cyclin-CDK interaction, we have constructed mutants in conserved residues of the N-terminal helix of Xenopus cyclins B2 and A1. The mutants showed altered binding affinities to Cdc2 and/or Cdk2. We also screened for mutations in the C-terminal lobe of CDK that exhibited different binding affinities for the cyclin-CDK complex. These mutations were at residues that interact with the cyclin N-terminal helix motif. The cyclin N-terminal helix mutations have a significant effect on the interaction between the cyclin-CDK complex and specific substrates, Xenopus Cdc6 and Cdc25C. These results suggest that the N-terminal helix of mitotic cyclins is required for specific interactions with CDKs and that to interact with CDK, specific substrates Cdc6 and Cdc25C require the CDK to be associated with a cyclin. The interaction between the cyclin N-terminal helix and the CDK C-terminal lobe may contribute to binding specificity of the cyclin-CDK complex.

Secretion of inhibin A and inhibin B throughout pregnancy and the early postpartum period in chimpanzees.

Plasma concentrations of inhibin A and inhibin B during pregnancy and early lactation in chimpanzees were determined by enzyme-linked immunosorbent assay (ELISA). Plasma samples were taken from five pregnant chimpanzees at 6-9, 10, 20 and 25 weeks of pregnancy, and following parturition. Throughout pregnancy and the early postpartum period, circulating inhibin A and inhibin B concentrations remained low, at similar levels to those during the normal menstrual cycle in chimpanzees. Concentrations of inhibin A in the placental homogenate were high enough to be measured by the ELISA and by bioassay, whereas circulating inhibin bioactivities in late pregnancy were too low to be measured. Plasma concentrations of FSH remained low with no significant changes throughout pregnancy and the postpartum period. Plasma concentrations of oestradiol-17beta and progesterone at 25 weeks of pregnancy were much higher than normal menstrual cycle levels. It was concluded that in chimpanzees the levels of circulating inhibin A and inhibin B remained low throughout pregnancy and the early postpartum period, and that the concentrations of bioactive dimeric inhibin did not increase towards the end of pregnancy. The suppression of circulating FSH levels during pregnancy is suggested to be controlled by steroid hormones that increased significantly in late pregnancy, and the present findings further suggest that the secretory pattern and role of inhibin during pregnancy in chimpanzees may be different from that in human and other primates.

Isolation and characterisation of a mutation in the PMR1 gene encoding a Golgi membrane ATPase, which causes hypersensitivity to over-expression of Clb3 in Saccharomyces cerevisiae.

We screened for mutant strains of Saccharomyces cerevisiae that are sensitive to overexpression of specific cyclins, and identified mutations in two genes that caused growth inhibition in response to mild overexpression of Clb3. One was the ANP1 gene, which encodes a glycosyltransferase previously identified by a similar strategy using Clb2 instead of Clb3. This paper describes the second strain of S. cerevisiae that is hypersensitive to Clb3 expression. The gene mutated in this strain was identified as PMR1, which encodes a Ca2+-ATPase located in the Golgi membrane. The protein product of pmr1-1 was truncated at residue 409 and thus lacked the C-terminal ATPase domain. The pmr1-1 strain was hypersensitive to over-expression of Clb3, but not Cln2, Clb5 or Clb2. The lethality due to Clb3 expression in pmr1-1 could be suppressed by adding Ca2+ ions to the medium. The pmr1-1 strain proved to be defective in glycosylation, and the defects in glycosylation were exacerbated by high levels of Clb3. On induction of Clb3 expression in the pmr1-1 strain, the cells arrested at anaphase with an elongated daughter bud. We discuss possible interpretations of this synthetic lethal phenotype.

Anti-apoptotic role of focal adhesion kinase (FAK). Induction of inhibitor-of-apoptosis proteins and apoptosis suppression by the overexpression of FAK in a human leukemic cell line, HL-60.

Focal adhesion kinase (FAK) has an anti-apoptotic role in anchorage-dependent cells via an unknown mechanism. To elucidate the role of FAK in anti-apoptosis, we have established several FAK cDNA-transfected HL-60 cell lines and examined whether FAK-transfected cells have resistance to apoptotic stimuli. FAK-transfected HL-60 (HL-60/FAK) cells were highly resistant to apoptosis induced with hydrogen peroxide (1 mm) and etoposide (50 microg/ml) compared with the parental HL-60 cells or the vector-transfected cells, when determined using viability assay, DNA fragmentation, and flow cytometry analysis. Because no proteolytic cleavage of pro-caspase 3 to mature caspase 3 fragment was observed in HL-60/FAK cells, FAK was presumed to inhibit an upstream signal pathway leading to the activation of caspase 3. HL-60/FAK activated the phosphatidylinositide 3'-OH-kinase-Akt survival pathway and exhibited significant activation of NF-kappaB with marked induction of inhibitor-of-apoptosis proteins (IAPs: cIAP-1, cIAP-2, XIAP), regardless of the hydrogen peroxide-treated or untreated conditions, whereas no significant IAPs were detected in the parental or vector-transfected HL-60 cells. Apoptotic agents induced higher NF-kappaB activation in HL-60/FAK cells than in HL-60/Vect cells, and it appeared that sustained NF-kappaB activation is critical to the anti-apoptotic states in HL-60/FAK cells. Mutagenesis of FAK cDNA revealed that Y397 and Y925, which are involved in the tyrosine-phosphorylation sites, were prerequisite for the anti-apoptotic activity as well as induction of IAPs, and that K454, which is involved in the kinase activity, was also required for the full anti-apoptotic activity of FAK. Taken together, we have demonstrated definitively that FAK-transfected HL-60 cells, otherwise sensitive to apoptosis, become resistant to the apoptotic stimuli. We conclude that FAK activates the phosphatidylinositide 3'-OH-kinase-Akt survival pathway with the concomitant activation of NF-kB and induction of IAPs, which ultimately inhibit apoptosis by inhibiting caspase-3 cascade.

Differential induction of JE/MCP-1 in subclones from a murine macrophage cell line, RAW 264.7: role of kappaB-3 binding protein.

The JE/MCP-1 gene is an immediate-early gene, and its product is a CC chemokine that attracts monocytes, basophils and T lymphocytes. JE/MCP-1 gene expression is induced by various inflammatory stimuli, but its transcriptional mechanism is not fully understood. To address this question, we obtained two subclones from a parental RAW264.7 cell line, one subline with low JE/MCP-1-producing capacity (named RAW.c11) and the other with high JE/MCP-1-producing capacity (named RAW.c25), in response to lipopolysaccharide (LPS). These subclones have no significant differences in CD14 expression, nitric oxide production, or production of other cytokines, including TNF-alpha or IL-1alpha/beta. In electrophoretic mobility shift assays (EMSA), there were no significant differences in DNA binding to the NF-kappaB-consensus sequence and interferon regulatory factor (IRF)-1,2 binding sequences. However, significantly higher binding activity to the NF-kappaB-like sequence (kappaB-3), which is located in the promoter region of the JE/MCP-1 gene, was shown by a high producer subclone than by a low producer subclone. Transient transfection analysis using deletion mutants of a 0.5-kb region from -467 to +59 identified an LPS-responsive region in a kappaB-3 site (from -169 to -132) in the high producer subclone. Mutation of this site markedly reduced sensitivity to LPS in the high producer subclone. These data suggest that a yet undefined nuclear factor may be involved in differential JE/MCP-1 gene transcription.

Identification of XDRP1; a Xenopus protein related to yeast Dsk2p binds to the N-terminus of cyclin A and inhibits its degradation.

Using the N-terminus of cyclin A1 in a two-hybrid screen as a bait, we identified a Xenopus protein, XDRP1, that contains a ubiquitin-like domain in its N-terminus and shows significant homology in its C-terminal 50 residues to Saccharomyces cerevisiae Dsk2 and Schizosaccharomyces pombe dph1. XDRP1 is a nuclear phosphoprotein in Xenopus cells, and its phosphorylation is mediated by cyclin A-dependent kinase. XDRP1 binds to both embryonic and somatic forms of cyclin A (A1 and A2) in Xenopus cells, but not to B-type cyclins. The N-terminal ubiquitin-like domain of XDRP1, but not the C-terminal Dsk2-like domain, is required for interaction with cyclin A. XDRP1 requires residues 130-160 of cyclin A1 for efficient binding, which do not include the destruction box of cyclin A. The addition of bacterially expressed XDRP1 protein to frog egg extract inhibited the Ca(2+)-induced degradation of cyclin A, but not that of cyclin B. The injection of XDRP1 protein into fertilized Xenopus eggs blocked embryonic cell division.

Maize genes specifically expressed in the outermost cells of root cap.

The cells at the periphery of the root cap are continuously sloughed off from the root into the mucilage, and are thought to be programmed to die. By using subtractive hybridization/differential screening and a transmembrane-domain trapping screening strategy involving expression screening in transfected COS-7 cells, we isolated two related maize cDNAs (ZmRCP1 and ZmRCP2) that are specifically expressed in the outermost one to three cells of the cap. ZmRCP1 and ZmRCP2 are homologous proteins of 37 kDa mature polypeptides with a region of regularly-spaced Cys residues and putative N-terminal signal peptides, and represent members of a novel protein family which is conserved among angiosperm and gymnosperm.

An altered nuclear migration into the daughter bud is induced by the cyclin A1-mediated Cdc28 kinase through an aberrant spindle movement in Saccharomyces cerevisiae.

A strain of Saccharomyces cerevisiae that contains an integrated copy of a Xenopus cyclin A1 gene under the control of the GAL1 promoter has been constructed. On inducing expression of cyclin A1, the nuclear migration that occurs prior to division becomes aberrant. Instead of migrating to the neck between the mother cell and daughter bud, the nucleus, the short mitotic spindle and its associated two spindle pole bodies entered the daughter bud. This phenotype was induced by expression of an indestructible cyclin mutant, but not by a mutated cyclin A1 unable to activate Cdc28 kinase. The nuclear abnormality induced by cyclin A1 was overcome by cdc28 mutations that abolish its ability to bind cyclin A1. Both yeast cyclin Clb3 and Xenopus mitotic cyclin B produced the same phenotype, whereas G1 cyclin Cln2 did not. The results suggest that the proper movement of the nucleus through the spindle function during mitosis requires the appropriate activity of Cdc28 kinase mediated by specific cyclins.

Fracture on removal of the ACE tibial nail.

We report four patients who sustained secondary fractures of the posterior wall of the tibial shaft during the removal of one pattern of intramedullary nail after fracture healing. The cause of this complication is discussed.

Xenopus cyclin A1 can associate with Cdc28 in budding yeast, causing cell-cycle arrest with an abnormal distribution of nuclear DNA.

BACKGROUND: Cyclins play a regulatory role in cell cycle progression, associated with cyclin-dependent kinases. We have investigated the structure-function relationships of cyclin A, mainly using Xenopus egg extracts in vitro. To further analyse the function and structure of cyclin A in vivo, we expressed Xenopus cyclin A1 in the budding yeast Saccharomyces cerevisiae. RESULTS: We herein show that vertebrate cyclin A1 can associate with endogenous Cdc28 to form histone H1 kinase. The growth of the yeast was inhibited by the expression of indestructible cyclin A1, but not by a non-Cdk binding cyclin A1 mutant. The induction of cyclin A1 expression in yeast caused cell cycle arrest with an abnormal distribution of nuclear DNA to the daughter bud. Suppressors of the cyclin A1-mediated growth arrest were identified as new alleles of the cdc28 mutation that reduced the binding of cyclin A1 and possessed different affinities for the cyclin-Cdc28 complexes. The temperature-sensitivity of the cdc28 mutation was thus preferentially suppressed by the endogenous cyclins CLN2 and CLB2. CONCLUSIONS: These results suggest that the Cdc28 protein kinase activity mediated by vertebrate cyclin A1 may be involved in the process of nuclear movement in the yeast, and thereby affect the dependence of the M phase on the completion of the S phase through a preferential binding affinity of the cyclin-Cdc28 complex.

FMLP actions and its binding sites in isolated human coronary arteries.

The chemoattractant f-Met-Leu-Phe (FMLP) can modulate human coronary arterial tone without the involvement of peripheral leukocytes. We investigated the actions of FMLP and its cellular mechanism in human coronary arteries isolated 2-3 h after death. A single dose of FMLP (0.01-10 microM) produced transient contraction (or, followed by relaxation) responses in most human coronary rings examined. These responses to FMLP were in large part mediated by the generation of cyclooxygenase products, mainly thromboxane A2 (TXA2) and prostaglandin I2 (PGI2). Radiolabeled N-formyl hexapeptide. 125I-f-Nle-Leu-Phe-Nle-Tyr-Lys bound densely to intimal and adventitial sites that accumulated macrophages (CD68-positive) with a Kd of 14-29 nM and, further, weakly to the media with a Kd of 2.4-3.6 microM. Several cell types including macrophages, endothelial cells and smooth muscle cells were positively immunostained for both TXA2 synthase and PGI2 synthase. However, there was no significant relation between the magnitude of the responses to FMLP and dense macrophage accumulation in the intimal plaques or the adventitia. A reverse transcription-polymerase chain reaction showed predominant expression of FMLP receptor homologues, FPRH1 and FPRH2 mRNA, in human coronary medial tissues relative to that in leukocytes. In conclusion. FMLP produced transient tension changes in human coronary arteries, mainly via the generation of TXA2 and PGI2. This effect of FMLP did not appear to be mediated by the activation of densely accumulated intimal and/or adventitial macrophages, but by the activation of unidentified medial tissue cells which might have functional FMLP receptor homologues.

[T cell receptor V beta repertoire of locoregional lymphocytes in malignant effusions].

We analyzed the T cell receptor (TCR) V beta repertoire of lymphocytes obtained from patients with malignant effusion treated by locoregional immunotherapy. Polymerase chain reaction using a panel of V beta subfamily specific oligonucleotide primers(V beta 1-20) was employed after reverse transcription of mRNA isolated from the locoregional cells. No major oligoclonality of TCR repertoire was observed in effusion lymphocytes before the treatment. The expression level of V beta 13.1 repertoire was significantly higher in effusion lymphocytes than in peripheral blood lymphocytes before treatment. V beta 20 gene expression increased significantly after locoregional administration of OK-432. It is suggested that TCR V beta 13.1 may be responsible for effusion lymphocytes and TCR V beta 20 for OK-432-related antigenic stimulus.

Locoregional immunotherapy of malignant ascites by intraperitoneal administration of OK-432 plus IL-2 in gastric cancer patients.

The clinical efficacy of intraperitoneal administration of OK-432 plus interleukin-2 (IL-2) for treating malignant ascites was evaluated in gastric cancer patients. Ten KE of OK-432 and 200,000 Jurkat units of IL-2 were intraperitoneally administered in tandem in the order given on alternate days at paracentesis. Of the 22 evaluable patients, 18 (81%) developed complete or partial responses, showing a cytologic disappearance of cancer cells and decrease of ascites. More than 50% of the patients obtained positive responses within 2 weeks after the initial administration of the drugs. Improvements of performance status and clinical symptoms such as abdominal fullness, followed by restoration of oral food intake and prolongation of survival time were observed in responders treated with OK-432 plus IL-2. Flow cytometric analysis demonstrated a predominant increase of the CD3+CD4+ cells, especially of the CD4+CD45RA- subset in the peritoneal cavity of the responders. Cytotoxicity assay after negative selection of the CD4+ cells with the antibody and complement revealed that the CD4+ subset possessed cytotoxic activity against autologous tumor cells. The results suggest that intraperitoneal administration of OK-432 plus IL-2 may not be only a practical but also an effective protocol for treating malignant ascites in gastric cancer patients.

Modulation of CD4 antigen expression on the lymphocyte surface by immunosuppressive acidic protein in cancer patients.

The immunomodulatory effect of human immunosuppressive acidic protein (IAP) on lymphocyte surface antigens was investigated. IAP inhibited lymphocyte responses to phytohemagglutinin in a dose-dependent manner. By flow cytometry, using fluorescein-isothiocyanate-labelled antibodies, the mean fluorescence intensity on peripheral blood lymphocytes (PBLs) decreased for CD4, slightly decreased for CD3 but showed no change for the CD8 and T cell receptor alpha-beta antigens in the presence of IAP. This CD4 antigen modulation by IAP was observed in PBLs freshly isolated from patients with unresectable cancer but not in those isolated from patients with resectable tumor or from healthy volunteers. The modulation of the CD4 antigen by IAP on the lymphocyte surface was correlated with an increment of serum IAP levels in cancer patients. The CD4 modulation could be induced in PBLs from healthy volunteers by culturing them with IAP in vitro. It is suggested that IAP may play a role in cancer-related immunosuppression through the down-modulation of the CD4 antigen on the lymphocyte surface.

[Skin reaction to OK-432 and its dosage for locoregional administration].

Establishment of optimal dosage of OK-432, a streptococcal preparation, was studied based on its skin test was studied. Locoregional immunotherapy using OK-432 was conducted for patients with malignant fluids. More OK-432 was administered to patients having a weaker skin reaction to OK-432 and less to those having a stronger one, corresponding to their redness diameter of skin reaction. There was a marked difference in OK-432-skin test among patients. Some patients with malignant fluids having small redness responded to the treatment after a large amount of OK-432, and others were well controlled by a smaller dosage of OK-432. It is suggested that OK-432-skin test may provide the optimal dosage for local treatment of malignant fluids.

Defective natural killer activity in gastric cancer patients: possible involvement of suppressor factor receptor.

The mechanism of defective natural killer (NK) activity in gastric cancer patients was studied with respect to the soluble immune suppressor factor receptor on NK cells. Phycoerythrin (PE)-conjugated anti-CD8 and -CD16 antibodies were used for the determination of NK phenotype, and fluorescein isothiocyanate (FITC)-conjugated wheat germ agglutinin (WGA) was for the lymphocyte surface receptor of soluble immune suppressor factor (SISF). It was found that NK activity of peripheral blood lymphocytes (PBLs) isolated from gactric cancer patients decreased with tumor progression in spite of an increasing tendency of their CD16+ population. The WGA+ population of PBLs inversely increased in parallel with cancer progression and a significant negative correlation was found between NK activity and the WGA+ population. Two-color flow cytometry showed a significant increase of WGA+ cells in the CD8dim and CD16+ population in advanced cancer patients. It is suggested that an increase of surface receptors for SISF on NK cells may, in part, cause the diminished NK activity in gastric cancer patients.