SFTG international collaborative study on in vitro micronucleus test IV. Using CHL cells

In this report, are presented the results of an international collaborative study on the in vitro micronucleus assay, using CHL cells. Fourteen laboratories participated in this study which was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Nine coded substances, having different modes of action and at different levels were assessed in the in vitro micronucleus test, using a common protocol. Mitomycin C was used as a positive control. In order to help to define a standard protocol on CHL cells, short and long treatment periods followed by various recovery times, with or without cytochalasin B, were compared. After an evaluation of the acceptability of the assays, the tested chemicals were classified as negative, positive or equivocal. Mannitol and clofibrate were judged as negative in all treatment schedules. Bleomycin was positive in all the treatment schedules, with an increase in the number of micronucleated cells in both mononucleate and binucleate cells when using cytochalasin B. This was also shown for the aneugens colchicine, diethylstilboestrol and griseofulvin, as expected. Urethane was judged as equivocal only after long treatment with cytochalasin B, and negative in all other treatment schedules. In any case, no genotoxic compound would have been missed with schedules including a short and a long treatment time, whether the treatment was followed by a recovery period or not and whether cytochalasin B was used or not. Thus, these results show that CHL cells were suitable for accurately detecting clastogenic and aneugenic compounds of various types in the in vitro micronucleus test.     

Blood flow responses in celiac and superior mesenteric arteries in the initial phase of digestion

Blood flow (BF) responses in the celiac artery (CA) and superior mesenteric artery (SMA) during and immediately after a meal are poorly understood. We characterized postprandial BF responses in these arteries in the initial phase of digestion. After a baseline measurement in the overnight fasting state, healthy subjects ingested solid food (300 kcal) and water ad libitum within 5 min (4.6 +/- 0.2 min, means +/- SE), and then rested for 60 min in the postprandial state. Mean blood velocities (MBVs) in CA (n = 7) and SMA (n = 9) and mean arterial pressure (MAP) were measured throughout the procedure. The MAP was divided by the MBV to yield the resistance index (RI). The MBV in CA and SMA started increasing within a minute after beginning the meal. The MBV in CA rapidly reached its peak increase (60 +/- 8% change from baseline) at 5 +/- 1 min after the start of the meal, whereas the MBV in SMA gradually reached its peak increase (134 +/- 14%) at 41 +/- 4 min after the start of the meal, reflecting a decrease in the RI for both CA and SMA. These findings suggested an earlier increase in CA and SMA MBV, implying that the increase of BF in some parts of the small intestine precedes the arrival of chyme.     

Partially Purified RhoA-Stimulated Phospholipase D Activity Specifically Binds to Phosphatidylinositol 4,5-Bisphosphate

Abstract: Phosphatidylinositol 4,5-bisphosphate (PIP₂) is absolutely required for the ADP-ribosylation factor-stimulated phospholipase D (PLD) activity. In the present study, partially purified rat brain PLD was found to be activated by another PLD activator, RhoA, when PIP₂, but not other acidic phospholipids, was included in vesicles comprising phosphatidylethanolamine (PE) and the PLD substrate phosphatidylcholine (PC) (PE/PC vesicles), demonstrating the absolute requirement of PIP₂ for the RhoA-stimulated PLD activation, too. It is interesting that the RhoA-dependent PLD activity in the partially purified preparation was drastically decreased after the preparation was incubated with and separated from PE/PC vesicles containing PIP₂. The PLD activity was extracted by higher concentrations of NaCl from the vesicles containing PIP₂ that were incubated with and then separated from the partially purified PLD preparation. These results demonstrate that RhoA-dependent PLD binds to PE/PC vesicles with PIP₂. The degree of binding of the RhoA-dependent PLD activity to the vesicles was totally dependent on the amount of PIP₂ in the vesicles and correlated well with the extent of the enzyme activation. Furthermore, it was found that a recombinant peptide of the pleckstrin homology domain of β-adrenergic receptor kinase fused to glutathione S-transferase, which specifically binds to PIP₂, inhibited the PIP₂-stimulated, RhoA-dependent PLD activity in a concentration-dependent manner. From these results, it is concluded that in vitro rat brain PLD translocates to the vesicles containing PIP₂, owing to its specific interaction with PIP₂, to access its substrate PC, thereby catalyzing the hydrolysis of PC. PLD appears to localize exclusively on plasma membranes of cells and tissues. An aminoglycoside, neomycin, that has high affinity for PIP₂ effectively extracted the RhoA-dependent PLD activity from rat brain membranes. This indicates that PIP₂ serves as an anchor to localize PLD on plasma membranes in vivo.     

Effect of piceatannol-rich passion fruit seed extract on human glyoxalase I–mediated cancer cell growth

Passion fruit seed extract (PFSE), a product rich in stilbenes such as piceatannol and scirpusin B, has various physiological effects. It is unclear whether PFSE and its stilbene derivatives inhibit cancer cell proliferation via human glyoxalase I (GLO I), the rate-limiting enzyme for detoxification of methylglyoxal. We examined the anticancer effects of PFSE in two types of human cancer cell lines with different GLO I expression levels, NCI–H522 cells (highly-expressed GLO I) and HCT116 cells (lowly-expressed GLO I). PFSE and its stilbenes inhibited GLO I activity. In addition, PFSE and its stilbenes supressed the cancer cell proliferation of NCI–H522 cells more than HCT116 cells. These observations suggest that PFSE can provide a novel anticancer strategy for prevention and treatment.     

Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin

We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.     

Integration of proteomic analyses to monitor the activity status of phosphorylation signaling

We performed here MS-based phosphoproteomics using both metal oxide affinity chromatography (pSTY proteomics) and anti-phosphotyrosine antibody (pY proteomics). The former method identified mainly phospho-serine and -threonine of nuclear or cytoplasmic proteins, whereas the latter did phosphotyrosine including more plasma membrane proteins and kinases. The overlap between these two methods was limited (24 tyrosine phosphorylation sites out of 325) and, by combining the two, coverage of the signaling molecules was enhanced as exemplified by Erk signaling. We also performed whole cell proteomics using an off-gel fractionator, and found 68.9% of the proteins identified by phosphoproteomics. Thus, the expression levels of phosphoproteins were roughly estimated. In addition to many uncharacterized phosphorylation sites, the dataset includes 136 sites that were experimentally verified elsewhere to be phosphorylated by a total of 83 kinases and kinase groups out of the 256 registered in the Phospho.ELM database. With the integration of various proteomic analyses and information from database, the responsible kinases of the identified phosphorylation sites and possibly their activity status were predicted by phosphorylation status and expression levels of their substrates, and thus our method may be able to monitor the activity status of phosphorylation signaling.