Follow-up testing of rodent carcinogens not positive in the standard genotoxicity testing battery: IWGT workgroup report

At the Plymouth Third International Workshop on Genotoxicity Testing in June 2002, a new expert group started a working process to provide guidance on a common strategy for genotoxicity testing beyond the current standard battery. The group identified amongst others "Follow-up testing of tumorigenic agents not positive in the standard genotoxicity test battery" as one subject for further consideration [L. Müller, D. Blakey, K.L. Dearfield, S. Galloway, P. Guzzie, M. Hayashi, P. Kasper, D. Kirkland, J.T. MacGregor, J.M. Parry, L. Schechtman, A. Smith, N. Tanaka, D. Tweats, H. Yamasaki, Strategy for genotoxicity testing and stratification of genotoxicity test results-report on initial activities of the IWGT Expert Group, Mutat. Res. 540 (2003) 177-181]. A workgroup devoted to this topic was formed and met on September 9-10, 2005, in San Francisco. This workgroup was devoted to the discussion of when it would be appropriate to conduct additional genetic toxicology studies, as well as what type of studies, if the initial standard battery of tests was negative, but tumor formation was observed in the rodent carcinogenicity assessment. The important role of the standard genetic toxicology testing to determine the mode of action (MOA) for carcinogenesis (genotoxic versus non-genotoxic) was discussed, but the limitations of the standard testing were also reviewed. The workgroup also acknowledged that the entire toxicological profile (e.g. structure-activity relationships, the nature of the tumor finding and metabolic profiles) of a compound needed to be taken into consideration before the conduct of any additional testing. As part of the meeting, case studies were discussed to understand the practical application of additional testing as well as to form a decision tree. Finally, suitable additional genetic toxicology assays to help determine the carcinogenic MOA or establish a weight of evidence (WOE) argument were discussed and formulated into a decision tree.     

Phenology, distribution, and host specificity of Solenopsis invicta virus-1

Studies were conducted to examine the phenology, geographic distribution, and host specificity of the Solenopsis invicta virus-1 (SINV-1). Two genotypes examined, SINV-1 and -1A, exhibited similar seasonal prevalence patterns. Infection rates among colonies of S. invicta in Gainesville, Florida, were lowest from early winter (December) to early spring (April) increasing rapidly in late spring (May) and remaining high through August before declining again in the fall (September/October). Correlation analysis revealed a significant relationship between mean monthly temperature and SINV-1 (p<0.0005, r=0.82) and SINV-1A (p<0.0001, r=0.86) infection rates in S. invicta colonies. SINV-1 was widely distributed among S. invicta populations. The virus was detected in S. invicta from Argentina and from all U.S. states examined, with the exception of New Mexico. SINV-1 and -1A were also detected in other Solenopsis species. SINV-1 was detected in Solenopsis richteri and the S. invicta/richteri hybrid collected from northern Alabama and Solenopsis geminata from Florida. SINV-1A was detected in S. geminata and Solenopsis carolinensis in Florida and the S. invicta/richteri hybrid in Alabama. Of the 1989 arthropods collected from 6 pitfall trap experiments from Gainesville and Williston, Florida, none except S. invicta tested positive for SINV-1 or SINV-1A. SINV-1 did not appear to infect or replicate within Sf9 or Dm-2 cells in vitro. The number of SINV-1 genome copies did not significantly increase over the course of the experiment, nor were any cytopathic effects observed. Phylogenetic analyses of SINV-1/-1A nucleotide sequences indicated significant divergence between viruses collected from Argentina and the U.S.     

Protein kinase Cdelta plays a non-redundant role in insulin secretion in pancreatic beta cells

Protein kinase C (PKC) is considered to modulate glucose-stimulated insulin secretion. Pancreatic beta cells express multiple isoforms of PKCs; however, the role of each isoform in glucose-stimulated insulin secretion remains controversial. In this study we investigated the role of PKCdelta, a major isoform expressed in pancreatic beta cells on beta cell function. Here, we showed that PKCdelta null mice manifested glucose intolerance with impaired insulin secretion. Insulin tolerance test showed no decrease in insulin sensitivity in PKCdelta null mice. Studies using islets isolated from these mice demonstrated decreased glucose- and KCl-stimulated insulin secretion. Perifusion studies indicated that mainly the second phase of insulin secretion was decreased. On the other hand, glucose-induced influx of Ca2+ into beta cells was not altered. Immunohistochemistry using total internal reflection fluorescence microscopy and electron microscopic analysis showed an increased number of insulin granules close to the plasma membrane in beta cells of PKCdelta null mice. Although PKC is thought to phosphorylate Munc18-1 and facilitate soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors complex formation, the phosphorylation of Munc18-1 by glucose stimulation was decreased in islets of PKCdelta null mice. We conclude that PKCdelta plays a non-redundant role in glucose-stimulated insulin secretion. The impaired insulin secretion in PKCdelta null mice is associated with reduced phosphorylation of Munc18-1.     

Production of Recombinant β-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta

Human β-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of α- and β-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using α-1,6-mannosyltransferase-deficient (och1Δ) yeast as the host. Genes encoding the α- and β-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (αα) and HexB (ββ). A total of 57 mg of β-hexosaminidase isozymes, of which 13 mg was HexA (αβ), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the β-subunit. The purified HexA was treated with α-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 ± 0.1 and 1.7 ± 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the β-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed.     

Efficient Extraction of Thioreodoxin from Saccharomyces cerevisiae by Ethanol

Thioredoxin, an antioxidant protein, is a promising molecule for development of functional foods because it protects the gastric mucosa and reduces the allergenicity of allergens. To establish a method for obtaining an ample amount of yeast thioredoxin, we found here that thioredoxin is released from Saccharomyces cerevisiae by treatment with 20% ethanol. We also found that Japanese sake contains a considerable amount of thioredoxin.     

Synthesis and antibacterial activities of new quinolone derivatives utilizing 1-azabicyclo[1.1.0]butane

The ring-opening reactions of 1-azabicyclo[1.1.0]butane 3 with thiols 6a-f gave 3-sulfenylazetidine derivatives 7a-f in 50-92% yields. Treatment of 3 with aromatic amines 11a-e and dibenzylamine 11f in the presence of Mg(ClO(4))(2) afforded the corresponding 3-aminoazetidine derivatives 12a-f in 24-53% yields. These azetidine derivatives were introduced into the C7 position of a quinolone nucleus 8 to afford the corresponding fluoroquinolones 9a-f and 13a-f in 21-83% yields. Some of them exhibited superior antibacterial activity against quinolone-susceptible MRSA in comparison with clinically used fluoroquinolones, such as levofloxacin, ciprofloxacin, and gatifloxacin.     

Identification of three protein disulfide isomerase members from Haemaphysalis longicornis tick

Three genes encoding putative protein disulfide isomerase (PDI) were isolated from the Haemaphysalis longicornis EST database and designed as HlPDI-1, HlPDI-2, and HlPDI-3. All three PDI genes contain two typical PDI active sites CXXC and encode putative 435, 499, and 488 amino acids, respectively. The recombinant proteins expressed in Escherichia coli all show PDI activities, and the activities were inhibited by a PDI-specific inhibitor, zinc bacitracin. Western blot analysis and real-time PCR revealed that three HlPDIs were present in all the developmental stages of the tick as well as in the midgut, salivary glands, ovary, hemolymph, and fatbody of adult female ticks, but the three genes were expressed at the highest level in the egg stage. HlPDI-1 is expressed primarily in the ovary and secondarily in the salivary glands. HlPDI-2 and HlPDI-3 are expressed primarily in the salivary gland, suggesting that the PDI genes are important for tick biology, especially for egg development, and that they play distinct roles in different tissues. Blood feeding induced significantly increased expression of HlPDI-1 and HlPDI-3 in both partially fed nymphs and adults. Babesia gibsoni-infected larval ticks expressed HlPDI-1 and HlPDI-3 2.0 and 4.0 times higher than uninfected normal larval ticks, respectively. The results indicate that HlPDI-1 and HlPDI-3 might be involved in tick blood feeding and Babesia parasite infection in ticks.     

Synthesis of fluorescent-labeled aeruginosin derivatives for high-throughput fluorescence correlation spectroscopy assays

The design and solid-phase synthesis of effective fluorescent-labeled aeruginosin derivatives and their application to the fluorescence correlation spectroscopy (FCS)-based competitive binding assay of an aeruginosin library are described. The phenolic hydroxyl group on the (R)-3-(4-hydroxyphenyl)lactic acid (d-Hpla) residue was observed to be suitable for connecting Rhodamine green derivative with minimum loss of biological activity. In addition, the FCS-based binding assay of the library using fluorescent-labeled chemical probes was also achieved.     

Synthesis and characterization of styrylchromone derivatives as beta-amyloid imaging agents

Several promising agents have been synthesized and evaluated for in vivo imaging probes of beta-amyloid plaques in Alzheimer's disease (AD) brain. Recently, we have developed flavone derivatives, which possess the basic structure of the 2-phenylchromone, as useful candidates for amyloid imaging agents. In an attempt to further develop novel tracers, we synthesized and evaluated a series of 2-styrylchromone derivatives, which replace the 2-phenyl substituent of flavone backbone with the 2-styryl. A series of radioiodinated styrylchromone derivatives were designed and synthesized. The binding affinities for amyloid plaques were assessed by in vitro binding assay using pre-formed synthetic Abeta(1-40) aggregates. The new series of styrylchromone derivatives showed high binding affinity to Abeta aggregates at the K(d) values of 32.0, 17.5 and 8.7nM for [(125)I]6, [(125)I]9, and [(125)I]12, respectively. In biodistribution studies using normal mice, [(125)I]6 and [(125)I]9 examined in normal mice displayed high brain uptakes with 4.9 and 2.8%ID/g at 2min post injection. The radioactivity washed out from the brain rapidly (1.6 and 1.0%ID/g at 60min post injection for [(125)I]6 and [(125)I]9, respectively). But [(125)I]12 did not show marked brain uptake, and the washout rate from the brain was relatively slow throughout the time course (1.1 and 1.4%ID/g at 2 and 30min post injection, respectively). Although additional modifications are necessary to improve the brain uptake and rapid clearance of non-specifically bound radiotracer, the styrylchromone backbone may be useful as a backbone structure to develop novel beta-amyloid imaging agents.