Ames test-negative carcinogen, ortho-phenyl phenol, binds tubulin and causes aneuploidy in budding yeast

Ortho-phenyl phenol (OPP) is broad-spectrum of fungicides and antibacterial agents. OPP tested negative in an Ames system and positive with respect to the formation of tumors in the urinary bladder in rats when administered in diet, showing attributes of an Ames test-negative carcinogen. It has also been demonstrated that OPP does not bind or cleave DNA in vivo or in vitro, rather dose-dependent protein binding in OPP-treated rats was observed. OPP, however, generates chromosomal aberrations including aneuploidy. Thus, the steps by which Ames test-negative carcinogens exert their effects need to be elucidated. Here, we used an assay of loss of heterozygosity (LOH) in Saccharomyces cerevisiae to determine the biological effects of OPP and its hepatic metabolite phenyl hydroquinone (PHQ). LOH was found to be induced by OPP and PHQ because of a functional chromosome loss: aneuploidy. PHQ bound to and interfered with the depolymerization of tubulin in vitro and arrested the cell-cycle at M and G1. These results indicate that OPP and PHQ damaged tubulin to cause mis-segregation of chromosome by delaying cell-cycle progression through mitosis, and as a consequence caused aneuploidy.     

Gain-of-function phenotypes of chemically synthetic CLAVATA3/ESR-related (CLE) peptides in Arabidopsis thaliana and Oryza sativa

Using 26 chemically synthetic CLAVATA3/ESR (CLE) peptides, which correspond to the predicted products of the 31 Arabidopsis CLE genes, we investigated the CLE peptide function in Arabidopsis and rice. Treatment with some CLE peptides inhibited root elongation in rice as well as in Arabidopsis. It also reduced the size of the shoot apical meristem in Arabidopsis but not in rice. Database searches revealed 47 putative CLE genes in the rice genome and multiple CLE domains in some CLE genes, indicating diverse CLE function in these plants.     

Biodegradation of bisphenol A and related compounds by Sphingomonas sp. strain BP-7 isolated from seawater

A bacterium capable of assimilating 2,2-bis(4-hydroxyphenyl)propane (bisphenol A), strain BP-7, was isolated from offshore seawater samples on a medium containing bisphenol A as sole source of carbon and energy, and identified as Sphingomonas sp. strain BP-7. Other strains, Pseudomonas sp. strain BP-14, Pseudomonas sp. strain BP-15, and strain no. 24A, were also isolated from bisphenol A-enrichment culture of the seawater. These strains did not degrade bisphenol A, but accelerated the degradation of bisphenol A by Sphingomonas sp. strain BP-7. A mixed culture of Sphingomonas sp. strain BP-7 and Pseudomonas sp. strain BP-14 showed complete degradation of 100 ppm bisphenol A within 7 d in SSB-YE medium, while Sphingomonas sp. strain BP-7 alone took about 40 d for complete consumption of bisphenol A accompanied by accumulation of 4-hydroxyacetophenone. On a nutritional supplementary medium, Sphingomonas sp. strain BP-7 completely degraded bisphenol A and 4-hydroxyacetophenone within 20 h. The strain degraded a variety of bisphenols, such as 1,1-bis(4-hydroxyphenyl)ethane, 2,2-bis(4-hydroxy-3-methylphenyl)propane, 2,2-bis(4-hydroxyphenyl)butane, and 1,1-bis(4-hydroxyphenyl)cyclohexane, and hydroxy aromatic compounds such as 4-hydroxyacetophenone, 4-hydroxybenzoic acid, catechol, protocatechuic acid, and hydroquinone. The strain did not degrade bis(4-hydroxyphenyl)methane, bis(4-hydroxyphenyl)sulfone, or bis(4-hydroxyphenyl)sulfide.     

In vitro inhibitory effects of Daphne oleoides ssp. oleoides on inflammatory cytokines and activity-guided isolation of active constituents

Aerial parts of Daphne oleoides Schreber ssp. oleoides (Thymelaeaceae) are used to treat rheumatoid arthritis and lumbago in Turkish folk medicine. In order to evaluate folkloric utilization, in vitro inhibitory effects of the ethyl acetate extract and fractions obtained from this extract on interleukin 1 (IL-1alpha, IL-1beta) and tumour necrosis factor (TNF-alpha) biosynthesis were studied. Through chemical isolation techniques and activity-guided fractionation process, seventeen compounds were isolated and their structures were elucidated (numbered 1-17). Diterpenoids genkwadaphnin (3) and 1,2-dehydrodaphnetoxin (6) and a coumarin derivative daphnetin (9) showed potent inhibitory activity and were found to be the main active ingredients. Furthermore, gnidilatin (4), gnidilatin-20 palmitate (5), genkwadaphnin-20-palmitate (7) and gnidicin-20-palmitate (8), having diterpenoid structure, and eudesmine (12), wikstromol (13) and matairesinol (14), having lignan structure, were determined to possess moderate inhibitory activity and may have a contributory role in the effect of the remedy.     

Design and synthesis of new orally active inhibitors of human neutrophil elastase

To identify new orally active inhibitors, further modification of 1 (ONO-6818) was performed. Peptidic derivatives 4b, 4c and 4n showed more potent inhibitory activity than nonpeptidic derivatives 3a-c. As a result, a series of peptidic inhibitors, 4a-s and 5a-v, were discovered. Among these N-aryl derivatives 5a-g, 5i, 5m and 5o-v showed oral activity. Their oral activity showed good correlation with their metabolic stability. Compounds 5h and 5j-l, which were extremely metabolically unstable in hamster plasma, did not show oral activity. Oral activity was considered to be determined by a combination of at least two factors: oral absorption and metabolic stability.     

Association of the physical and chemical properties and the cytotoxicity of metal oxide nanoparticles: metal ion release, adsorption ability and specific surface area

Association of cellular influences and physical and chemical properties were examined for 24 kinds of industrial metal oxide nanoparticles: ZnO, CuO, NiO, Sb(2)O(3), CoO, MoO(3), Y(2)O(3), MgO, Gd(2)O(3), SnO(2), WO(3), ZrO(2), Fe(2)O(3), TiO(2), CeO(2), Al(2)O(3), Bi(2)O(3), La(2)O(3), ITO, and cobalt blue pigments. We prepared a stable medium dispersion for each nanoparticle and examined the influence on cell viability and oxidative stress together with physical and chemical characterizations. ZnO, CuO, NiO, MgO, and WO(3) showed a large amount of metal ion release in the culture medium. The cellular influences of these soluble nanoparticles were larger than insoluble nanoparticles. TiO(2), SnO(2), and CeO(2) nanoparticles showed strong protein adsorption ability; however, cellular influences of these nanoparticles were small. The primary particle size and the specific surface area seemed unrelated to cellular influences. Cellular influences of metal oxide nanoparticles depended on the kind and concentrations of released metals in the solution. For insoluble nanoparticles, the adsorption property was involved in cellular influences. The primary particle size and specific surface area of metal oxide nanoparticles did not affect directly cellular influences. In conclusion the most important cytotoxic factor of metal oxide nanoparticles was metal ion release.     

Comprehensive royal jelly (RJ) proteomics using one- and two-dimensional proteomics platforms reveals novel RJ proteins and potential phospho/glycoproteins

Royal jelly (RJ) is an exclusive food for queen honey bee (Apis mellifera L.) that is synthesized and secreted by young worker bees. RJ is also widely used in medical products, cosmetics, and as health foods. However, little is known about RJ functionality and the total protein components, although recent research is attempting to unravel the RJ proteome. We have embarked on a detailed investigation of the RJ proteome, using a modified protein extraction protocol and two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with tandem mass spectrometry. Simultaneously, we examined total soluble protein from RJ collected at 24, 48, and 72 h after honey bee larvae deposition twice (in two flower blooming seasons), to check differences, if any, in RJ proteome therein. Both 1- and 2-D gels stained with silver nitrate revealed similar protein profiles among these three time points. However, we observed a clear difference in two bands (ca. MW of 55 and 75 kDa) on 1-D gel between the first and the second collection of RJ. A similar difference was also observed in the 2-D gel. Except for this difference, the protein profiles were similar at the 3 time points. As the RJ from 48 (or sometimes 72) is commercially used, we selected the RJ sample at 48 h for detailed analysis with the first collection. 1-DGE identified 90 and 15 proteins from the first and second selection, respectively; in total, 47 nonredundant proteins were identified. 2-DGE identified 105 proteins comprising 14 nonredundant proteins. In total, 52 nonredundant proteins were identified in this study, and other than the major royal jelly protein family and some other previously identified proteins, 42 novel proteins were identified. Furthermore, we also report potentially post-translationally modified (phosphorylation and glycosylation) RJ proteins based on the Pro-Q diamond/emerald phosphoprotein/glycoprotein gel stains; MRJP 2p and 7p were suggested as potential phosphoproteins. The 2-DGE data were integrated to develop a 2-D gel reference map, and all data are accessible through RJ proteomics portal (http://foodfunc.agr.ibaraki.ac.jp/RJP.html).     

Systematic investigation of the hemolymph proteome of Manduca sexta at the fifth instar larvae stage using one- and two-dimensional proteomics platforms

Manduca sexta is an excellent insect model for studying insect physiology, including hemolymph proteins. Larvae stages of this insect are highly damaging to tobacco leaves causing a drastic decrease in crop yield. Investigation on the larval biology should help in controlling its destructive potential, thus increasing crop yields. The hemolymph is the source of its immunity to disease and environmental factors, which invariably involves protein components. To better understand the physiology of M. sexta and the protein components expressed during its life cycle, two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with N-terminal amino acid sequencing and liquid chromatography-mass spectrometry, were employed to analyze the fifth instar larvae hemolymph proteins. These proteomics approaches together identified 123 proteins, which constituted a total of 58 nonredundant proteins and belonged to 10 functional categories. Defense (49%), transport and metabolism (15%), storage (9%), and metamorphosis (7%) categories were highly represented accounting for 80% of the identified proteins. Besides identification of previously reported proteins, 18 novel proteins were identified, which include the lipoprotein-releasing system transmembrane protein lolC, 50S ribosomal protein L24, inducible serine protease inhibitor 1, imaginal disk growth factor, protein disulfide-isomerase-like protein ERp57, etc. The 2-DGE data were integrated to develop a 2-D gel reference map. Data obtained from 1-DGE and 2-DGE analyses are accessible through the M. sexta proteomics portal ( http://foodfunc.agr.ibaraki.ac.jp/mansehemoprot.html). Together, this study provides evidence for the presence of a large number of functionally diverse protein families in the hemolymph of M. sexta. These proteins correlate well with the fifth instar stage, the transition from larvae to pupae.     

Urinary interleukin-2 may predict clinical outcome of intravesical bacillus Calmette-Guérin immunotherapy for carcinoma in situ of the bladder

PURPOSE: The mechanism by which bacillus Calmette-Guérin (BCG) mediates antitumor activity has not been clearly established. Specific cytokines in the urine after BCG intravesical instillation therapy may serve as a prognostic factor of treatment response. In this study, various urinary cytokines such as interleukin-1beta (IL-1beta), IL-2, IL-6, IL-8. IL-10, IL-12, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) were measured. MATERIALS AND METHODS: In total 20 patients were treated with BCG intravesical instillation therapy for carcinoma in situ of the bladder. At the completion of the first and eighth instillations, spontaneously voided urine specimens were collected before BCG instillation, every 2 h until 12 h, and thereafter until 24 h. All specimens were ultrafiltrated using an ADVANTEC UK-10 membrane. The cytokines were measured using ELISA and RIA techniques. RESULTS: Significantly higher levels of IL-2, IL-6, IL-8, IL-10, IFN-gamma, and TNF-alpha were detected in the eighth instillation as compared to the first instillation ( p<0.001). After BCG intravesical instillation therapy, treatment failure occurred in 6 of the 20 patients (30%), including primary failure (persistence of CIS) in 3, and de novo failure (tumor recurrence) in 3 with a median follow-up of 46.9 months. Significantly higher production of IL-2, IL-6, IL-8, IL-10, and TNF-alpha was observed in the responder group than in the non-responder group ( p<0.05). Multivariate analysis revealed IL-2 as an independent prognostic cytokine of responder status. CONCLUSIONS: This study indicates that urinary IL-2 at the eighth instillation of BCG may serve as a valuable prognostic factor of treatment efficacy as well as tumor recurrence after treatment.