Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin‐dependent neutrophil lysis

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA‐treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA‐treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA‐induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.     

Neutrophils become refractory to phorbol myristate acetate when treated with Ca2+-ionophore and Ca2+

Human neutrophils deprived of divalent cations by treatment with ionophore A23187 in the presence of ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) showed superoxide release when they were preincubated with calcium and then treated with the ionophore. The release was not observed when the ionophore was added first and then calcium was added more than 5 min later. The absence of the release in this case can be ascribed to a refractoriness of the cells to stimuli, because the cells did not release superoxide on stimulation with phorbol myristate acetate (PMA). The cells pretreated with either calcium or the ionophore alone did release superoxide on addition of PMA. The refractoriness of the cells to PMA depended on the concentrations of calcium and the ionophore and on the time interval between the two treatments. Calcium could be replaced with Cd2+ but not with Mg2+, Ba2+, or Sr2+. The release of granular enzymes was observed when the depleted cells were pretreated with the ionophore and then with calcium. These observations indicate that calcium has dual effects on the superoxide release of neutrophils, i.e., it stimulates the cells and makes them refractory to stimuli, depending on the time interval after the addition of the ionophore, and it also regulates the enzyme release by a different mechanism.     

Effect of Intracellular Glutathione Level on the Production of 6-Methoxymellein in Cultured Carrot (Daucus carota) Cells

To produce phytoalexin, 6-methoxymellein (6-MM) was induced in suspension cultures of carrot (Daucus carota) by buthionine sulfoximine (BSO) and CuCl2. Addition of BSO (a specific inhibitor of glutathione [GSH] synthesis) to the cultures lowered the cellular GSH levels. This depletion of GSH was BSO-concentration dependent, and the extent of 6-MM accumulation was dependent on the GSH depletion. The accumulation of 6-MM induced by BSO was suppressed by exogenous GSH. Exogenous H2O2 stimulated the production of 6-MM when added 1 d after BSO treatment, whereas H2O2 added at time zero or on the 4th d of BSO treatment did not. Moreover, a synergistic effect of simultaneous addition of BSO and CuCl2 was observed. These results suggest that active oxygen species may be involved in the triggering of 6-MM synthesis.     

Metabolic pattern of polymorphonuclear leucocytes induced by trypsin-digested microsomes.

The addition of trypsin [EC 3.4.21.4]-digested liver microsimes induced cyanideinsensitive respiration in guinea pig polymorphonuclear leucocytes with concomitant acceleration of the hexose monophosphate oxidative pathway. The respiration was insensitive to inhibitors of mitochondrial respiration but sensitive to glycolytic inhibitors. These metabolic alterations are similar to those associated with phagocytosis, though the digested mocrosomes were apparently not taken up by the cells and prpbably trigger the netabolic changes by interaction with the cellular membrane. Intact microsomes or microsomes treated with chymotrypsin [EC 3.4.21.1], bacterial proteinase, ribonuclease [EC 3.1.4.22], or neuraminidase [EC 3.2.1.18] could not induce such respiration.     

Simvastatin-romidepsin combination kills bladder cancer cells synergistically

The HMG-CoA reductase inhibitor simvastatin activates AMP-activated protein kinase (AMPK) and thereby induces histone acetylation. We postulated that combining simvastatin with the histone deacetylase (HDAC) inhibitor romidepsin would kill bladder cancer cells by inducing histone acetylation cooperatively. The combination of romidepsin and simvastatin induced robust apoptosis and killed bladder cancer cells synergistically. In murine subcutaneous tumor models using MBT-2 cells, a 15-day treatment with 0.5 mg/kg romidepsin and 15 mg/kg simvastatin was well tolerated and inhibited tumor growth significantly. Mechanistically, the combination induced histone acetylation by activating AMPK. The combination also decreased the expression of HDACs, thus further promoting histone acetylation. This AMPK activation was essential for the combination's action because compound C, an AMPK inhibitor, suppressed the combination-induced histone acetylation and the combination's ability to induce apoptosis. We also found that the combination increased the expression of peroxisome proliferator-activated receptor (PPAR) γ, leading to reactive oxygen species production. Furthermore, the combination induced endoplasmic reticulum (ER) stress and this ER stress was shown to be associated with increased AMPK expression and histone acetylation, thus playing an important role in the combination's action. Our study also suggests there is a positive feedback cycle between ER stress induction and PPARγ expression.     

Urinary and plasma proteomics to discover biomarkers for diagnosing between diabetic nephropathy and minimal change nephrotic syndrome or membranous nephropathy

The choice of treatment for primary nephrotic syndrome depends on the pathologic type of the disorder. Renal biopsy is necessary for a definitive diagnosis, but it is burdensome for the patients, and can be avoided if tests could be performed using urine or plasma. In this study, we analyzed 100 urinary proteins, 141 plasma proteins, and 57 urine/plasma ratios in cases of diabetic nephropathy (DN; n = 11), minimal change nephrotic syndrome (MCNS; n = 14), and membranous nephropathy (MN; n = 23). We found that the combination of urinary retinol-binding protein 4 and SH3 domain-binding glutamic acid-rich-like protein 3 could distinguish between MCNS and DN, with an area under the curve (AUC) of 0.9740. On the other hand, a selectivity index (SI) based on serotransferrin and immunoglobulin G, which is often used in clinical practice, distinguished them with an AUC of 0.9091. Similarly, the combination of urinary afamin and complement C3 urine/plasma ratio could distinguish between MN and DN with an AUC of 0.9842, while SI distinguished them with an AUC of 0.8538. Evidently, the candidates identified in this study were superior to the SI method. Thus, the aim was to test these biomarkers for accurate diagnosis and to greatly reduce the burden on patients.     

Effect of hypo-osmotic environmental changes on the expression of gonadotropin-releasing hormone,its receptor,and gonadotropin hormone subunit mRNA in adult chum salmon (Oncorhynchus keta)

Migrating fish such as salmonids are affected by external environmental factors and salinity changes are particularly important, influencing spawning migration. The aim of this study was to test whether changes in salinity would affect the expression of the hypothalamic-pituitary-gonadal (HPG) axis hormones (gonadotropin-releasing hormones (GnRHs) [salmon GnRH and chicken GnRH-II], GnRH receptors [GnRHR1 and GnRHR5], and mRNA of the gonadotropin hormone [GTH] subunits [GTHα, follicle stimulating hormone β, and luteinizing hormone β]) in chum salmon (Oncorhynchus keta). Fish were progressively transferred from seawater (SW) through 50% SW to freshwater (FW), and the relationship between the osmoregulatory hormone prolactin (PRL) and sexual maturation was determined. The expression and activity of HPG hormones and their receptors, and levels of estradiol-17β and PRL increased after fish were transferred to FW, demonstrating that changes in salinity stimulate the HPG axis and PRL production in migrating chum salmon. These findings reveal details about the role of the endocrine system in maintaining homeostasis and stimulating sexual maturation and reproduction in response to salinity changes in this species.