K252a: A new blocker of the cell-cycle at G1 phase in a human hepatoma cell line

The administration of 200 nM K252a to HuH7 suppressed the proliferation of the cells almost completely. The uptake of [³H]thymidine was inhibited, and flow cytometry revealed only one peak at 2C on day 3 after treatment with 100 nM K252a. The expression of proto-oncogene c-myc was not reduced. Despite the blockage at G1, both the size of the cells and the amount of cell protein had increased by 4 times by day 3 after treatment with K252a, while the cells secreted albumin and -fetoprotein into the medium as usual. These results show that K252a can increase the cell size of HuH7 without losing its function by blocking the cell cycle at G1 phase.     

Administration of D-alanine did not cause increase of D-amino acid oxidase activity in mice.

D-amino acid oxidase (DAAO) activity was not altered in the liver and kidney by oral administration of D-alanine to adult mice. The enzyme was apparently not induced by the enteric microflora either, since the enzyme activity in the liver and kidney of germ-free mice was not different from that of specific-pathogen-free mice. The times of appearance of DAAO activity and of free D-amino acids in the kidney were elucidated using suckling mice. DAAO activity started to increase 7 days after birth, and reached almost the adult level by 28 days. The content of free neutral D-amino acids also increased with age, in a similar fashion. A possible conclusion is that the enzyme activity normally increases during this period, to eliminate the free D-amino acids which have increased with age in the suckling mice. Consequently, the administration of D-alanine had no further effect in increasing enzyme activity.     

Administration of D-alanine did not cause increase of D-amino acid oxidase activity in mice

D-amino acid oxidase (DAAO) activity was not altered in the liver and kidney by oral administration of D-alanine to adult mice. The enzyme was apparently not induced by the enteric microflora either, since the enzyme activity in the liver and kidney of germ-free mice was not different from that of specific-pathogen-free mice. The times of appearance of DAAO activity and of free D-amino acids in the kidney were elucidated using suckling mice. DAAO activity started to increase 7 days after birth, and reached almost the adult level by 28 days. The content of free neutral D-amino acids also increased with age, in a similar fashion. A possible conclusion is that the enzyme activity normally increases during this period, to eliminate the free D-amino acids which have increased with age in the suckling mice. Consequently, the administration of D-alanine had no further effect in increasing enzyme activity.     

Modulation of C2 and C3 gene expression of human peripheral blood monocytes by interleukin 1 beta, interferon gamma, tumor necrosis factor alpha and lipopolysaccharide.

The effect of interleukin 1 beta (IL-1 beta), interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF alpha) and lipopolysaccharide (LPS) on the expression of the C2 and C3 genes in human adherent monocytes was studied. Stimulation of monocytes with IFN-gamma increased both C2 and C3 mRNA. IL-1 beta also increased C2 mRNA level, whereas C3 gene expression was not enhanced. TNF alpha failed to increase either C2 or C3 mRNA. LPS increased C2 mRNA, but suppressed C3 gene expression. These results suggest that C2 and C3 production by monocytes is regulated by IL-1 beta and IFN-gamma in the local tissues.     

siRNA-mediated knock-down of NOX3: therapy for hearing loss?

Cisplatin is a widely used chemotherapeutic agent that causes significant hearing loss. Previous studies have shown that cisplatin exposure is associated with increase in reactive oxygen species (ROS) in the cochlea. The inner ear expresses a unique isoform of NADPH oxidase, NOX3. This enzyme may be the primary source of ROS generation in the cochlea. The knockdown of NOX3 by pretreatment with siRNA prevented cisplatin ototoxicity, as demonstrated by preservation of hearing thresholds and inner ear sensory cells. Trans-tympanic NOX3 siRNA reduced the expression of NOX3 and biomarkers of cochlear damage, including transient receptor vanilloid 1 (TRPV1) channel and kidney injury molecule-1 (KIM-1) in cochlear tissues. In addition, siRNA against NOX3 reduced apoptosis as demonstrated by TUNEL staining, and prevented the increased expression of Bax and abrogated the decrease in Bcl2 expression following cisplatin administration. Trans-tympanic administration of siRNA directed against NOX3 may provide a useful method of attenuating cisplatin ototoxicity. In this paper, we review recent publications dealing with the role of NOX3 in ototoxicity and the effects of siRNA against cisplatin-induced hearing loss.     

Modulation of C2 and C3 gene expression of human peripheral blood monocytes by interleukin 1β, interferon γ, tumor necrosis factor α and lipopolysaccharide

The effect of interleukin 1β (IL-1β), interferon γ (IFN-γ), tumor necrosis factor α (TNFα) and lipopolysaccharide (LPS) on the expression of the C2 and C3 genes in human adherent monocytes was studied. Stimulation of monocytes with IFN-γ increased both C2 and C3 mRNA. IL-1β also increased C2 mRNA level, whereas C3 gene expression was not enhanced. TNFα failed to increase either C2 or C3 mRNA. LPS increased C2 mRNA, but suppressed C3 gene expression. These results suggest that C2 and C3 production by monocytes is regulated by IL-1β and IFN-β in the local tissues.     

Interaction of salicylate and noise results in mortality of rats

Summary Survival as a function of salicylate dose and the intensity of environmental noise was investigated in 150 adult female pigmented rats. Rats were assigned to groups (n=6/group) defined by combinations of salicylate levels from 0- (saline) to 300 mg/kg, injected subcutaneously, and noise levels from ambient noise to 98 dB SPL, presented daily for 10-h periods for up to 17 days. Mortality occurred in groups exposed to the higher combinations of salicylate and noise.     

Nitric oxide mediates histamine induced down-regulation of H<Subscript>2</Subscript> receptor mRNA and internalization of the receptor protein (R1)

During agonist-dependent long-term stimulation of cells, histamine receptor subtypes are frequently down-regulated. However, the mechanisms underlying the modulation of receptor expression during long-term histamine stimulation have yet to be resolved. Based on our recently reported results showing an H₁-mediated down-regulation of histamine H₂ receptor mRNA in endothelial cells, our aim was to characterize the mechanism controlling rapid and long-term histamine-mediated modulation of H₂ receptor expression in more detail. We were able to show that the histamine-induced down-regulation of H₂ receptor mRNA and cell surface expression lasting for 24 h was accompanied by augmentation of the receptor protein level in the cytoplasmatic fraction of endothelial cells for this time period. Furthermore, changes in receptor protein levels in whole-cell lysate were negligible, indicating that the rapid and prolonged modulation of cell surface H₂ receptor levels by histamine was regulated solely via internalization. The role of nitric oxide (NO) as a key mediator in histamine-stimulated cell responses was underlined by subsequent studies showing the attenuation of histamine-induced H₂ receptor mRNA down-regulation and protein trafficking following NO synthase isozyme inhibition.Received 11 March 2003; received after revision 11 June 2003; accepted 17 June 2003     

pEGFPN1-dnEGFR对胃癌细胞的化疗增敏作用

目的研究人 EGFR显性负性突变体真核表达载体(pEGFPN1 dnEGFR)对人胃癌细胞株 SGC 7901和 NCI N87化疗敏感性的影响,并探讨其可能机制.方法 MTT法测定奥沙利铂对稳定转染 pEGFPN1 dnEGFR和 pEGFP N1载体的两种胃癌细胞的量效反应.奥沙利铂作用各组细胞24h后,RT PCR检测各组细胞中 Caspase 3和 CyclinD1的 mRNA表达情况;Westernblot检测各组细胞中 Caspase 3和 CyclinD1蛋白表达情况.结果转染 pEGFPN1 dnEGFR后,两种胃癌细胞对奥沙利铂的敏感性增加,奥沙利铂对 pEGFPN1 dnEGFR转染组细胞的增殖抑制率(VI)与对照组相比有显著提高(P<0.05).RT PCR显示 pEGFPN1 dnEGFR转染组细胞 CyclinD1mRNA表达较对照组下降,而 Caspase 3mRNA表达较对照组升高(P<0.05);Westernblot显示 pEGFPN1 dnEG FR转染组细胞 CyclinD1蛋白表达较对照组下降,而 Caspase 3蛋白表达较对照组升高(P<0.05).结论 EGFR显性负性突变体能提高胃癌细胞对化疗药物奥沙利铂的敏感性,其机制可能与 Caspase 3和 CyclinD1有关     

Protein kinase-dependent overexpression of the nuclear protein pirin in c-JUN and RAS transformed fibroblasts

Signalling via the protein kinase Raf-MEK-ERK pathway is of major importance for transformation by oncogenes. To identify genes affected by inhibition of this pathway, c-JUN transformed rat fibroblasts were treated with a MEK1 inhibitor (PD98059) and subjected to two-dimensional gel electrophoresis after cell lysis. Gene products with expression influenced by MEK1 inhibition were determined by mass spectrometry of fragments from in-gel tryptic digestions. The expression of pirin, a nuclear factor I-interacting protein, was lowered after inhibition of MEK1. Western blot analysis revealed increased expression of pirin in RAS and c-JUN transformed cells in the absence of PD98059. Inhibition of MEK1 also led to reduced expression of α-enolase, phosphoglycerate kinase, elongation factor 2 and heterogeneous nuclear ribonucleoprotein A3, the latter two being detected as truncated proteins. In contrast, the level of ornithine aminotransferase was increased. We conclude that inhibition of MEK1 results in major alterations of protein expression in c-JUN transformed cells, suggesting that this pathway is important for oncogene-induced phenotypic changes. Received 30 December 1998; accepted 12 January 1999     

Alleviation of mortality induced by salicylate and stress

Protection from the deleterious effects of the interaction of environmental stress and salicylate by calcium supplement was investigated in 96 pigmented rats. Within a 2×2×4 factorial design, rats were assigned to groups defined by:A) ad lib access to 1) plain tap water, or 2) 50 mM calcium chloride solution;B) exposure to stressors consisting of daily 10 h periods of 1) 98 dB SPL noise, or 2) confinement precluding movements;C) daily injections of 233, 350, or 410 mg/kg of sodium salicylate or the saline vehicle. For subjects maintained on tap water, weight loss and mortality increased with salicylate levels, with all subjects dying in the group drinking water and injected with 410 mg/kg. Calcium protected all of the subjects in the noise stress group but not in the confined group.     

Dexamethasone enhances CTLA-4 expression during T cell activation

T cell activation is enhanced by the costimulatory interaction of B7 on antigen-presenting cells and CD28 on T cells, resulting in long-term T cell proliferation, differentiation and production of large amounts of cytokines, such as interleukin (IL)-2. CTLA-4 is a co-stimulation receptor that shares 31% homology with CD28 and binds B7 family members with higher affinity. CTLA-4 is transiently expressed intracellularly and on the cell surface following activation of T cells. We have studied the kinetics of CTLA-4 expression and the effects of dexamethasone on CTLA-4 expression during T cell activation in cultures of mouse spleen cells stimulated by a mixture of immobilized anti-CD3 and anti-CD28 monoclonal antibodies (anti-CD3/CD28 mAb) or concanavalin A (ConA). CTLA-4 expression peaked on day 2 and returned to background levels after 7 days. Dexamethasone was found to potentiate CTLA-4 expression in a dose-dependent manner with an EC₅₀ effective concentration 50%) of about 10⁻⁸ M. In contrast, other immunosuppressive agents, such as rapamycin or cyclosporin A had no or an inhibitory effect on CTLA-4 expression, respectively. Dexamethasone also stimulated CD28 expression, but inhibited IL-2R expression during anti-CD3/CD28 mAb-induced mouse splenic T cell activation. Western blot analyses of lysates of activated mouse T cells showed that dexamethasone increased CTLA-4 protein levels twofold during anti-CD3/CD28 mAb-induced activation. Dexamethasone also enhanced CTLA-4 messenger RNA twofold as quantified by ribonuclease protection assay. The effects of dexamethasone on CTLA-4 expression were glucocorticoid-specific and completely inhibited by the glucocorticoid receptor antagonist mifepristone (RU486), indicating that the effect of dexamethasone on CTLA-4 expression is mediated through the glucocorticoid receptor. In conclusion, the immunosuppressive agent dexamethasone actually stimulates CTLA-4 expression, which is involved in downregulation of T cell activation. Received 19 May 1999; received after revision 13 July 1999; accepted 13 July 1999     

The effects of salicylate and aspirin on the activity of phosphorylase a in perfused hearts of rats

Summary Sodium salicylate and aspirin are known to have a glycogenolytic effect as judged by either the glycogen level or lactate production in perfused hearts of rats. In this work it was possible to demonstrate that phosphorylase a level was increased in the hearts subjected to the action of these drugs.     

Testosterone derivatives are neuroprotective agents in experimental diabetic neuropathy

In this study we have assessed the effect of testosterone (T), dihydrotestosterone (DHT) and 5αandrostan-3α, 17β-diol (3α-diol) therapies on diabetic neuropathy. Diabetes was induced in adult male rats by the injection of streptozotocin and resulted in decreased T and increased 3α-diol levels in plasma and in decreased levels of pregnenolone and DHT in the sciatic nerve. Moreover, a reduced expression of the enzyme converting Tinto DHT (i.e., the 5α-reductase) also occurs at the level of sciatic nerve, suggesting that the decrease of DHT levels could be due to an impairment of this enzyme. Chronic treatment for 1 month with DHT or 3α-diol increased tail nerve conduction velocity and partially counteracted the increase of thermal threshold induced by diabetes. Treatment with DHT increased tibial Na⁺,K⁺-ATPase activity and the expression of myelin protein P0 in the sciatic nerve.DHT, 3α-diol and T reversed the reduction of intra-epidermal nerve fiber density induced by diabetes. These observations indicate that T metabolites can reverse behavioral, neurophysiological, morphological and biochemical alterations induced by peripheral diabetic neuropathy. I. Roglio, R. Bianchi: These authors contributed equally to this study. Received 4 January 2007; received after revision 13 February 2007; accepted 27 March 2007     

Cyclin D3 and p53 mediate sulforaphane-induced cell cycle delay and apoptosis in non-transformed human T lymphocytes

Despite experimental evidence that sulforaphane can exert chemopreventive effects, whether these effects are specific for neoplastic cells is not known. Following our previous demonstration that sulforaphane induces cell cycle arrest and apoptosis in human T lymphoblastoid Jurkat leukemia cells and increases p53 and bax protein expression, we tested sulforaphane on non-transformed phytohemagglutinin-stimulated human lymphocytes. Here, we demonstrate that sulforaphane arrested cell cycle progression in G, phase, through a decrease in the protein expression of cyclin D3. Moreover, sulforaphane induced apoptosis (and also necrosis), mediated by an increase in the expression of p53. These findings suggest that sulforaphane is a growth modulator for T cells. Our in vitro evidence that sulforaphane is active and even cytotoxic in normal as well as transformed lymphocytes raises important questions regarding its suitability for cancer chemoprevention.     

Identification of factor XIII-A as a marker of alternative macrophage activation

Factor XIII subunit A of blood coagulation (FXIII-A) is known to be synthesized but not secreted by the monocyte/macrophage cell line. On the basis of its intracellular localization and substrate profile, FXIII-A is thought to be involved in certain intracellular processes. Our present study was designed to monitor the changes in FXIII-A gene expression and protein production in long-term culture of human monocytes during their differentiation into macrophages in the presence of activating agents (interleukin-4, interferon-γ, Mycobacterium bovis BCG) inducing classical and alternative activation pathways. By using quantitative RT-PCR and fluorescent image analysis at the single-cell level we demonstrated that the expression of FXIII-A both at the mRNA as well as at the protein level is inversely regulated during the two activation programmes. Here we conclude that FXIII-A expression is an intracellular marker for alternatively activated macrophages, while its absence in monocyte-derived macrophages indicates their classically activated state.Received 2 June 2005; received after revision 12 July 2005; accepted 22 July 2005     

Effects of long-term feeding of glibenclamide on normal rats

Summary Prolonged administration of glibenclamide decreased blood sugar, liver glycogen and protein and increased liver and serum lipids and organic phosphates of liver in normal rats. A significant weight increase observed in glibenclamide group of rats is attributed to lipid accumulation.