Initiation of Genome Instability and Preneoplastic Processes through Loss of Fhit Expression

Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability.     

Novel In Vivo Imaging Analysis of an Inner Ear Drug Delivery System in Mice: Comparison of Inner Ear Drug Concentrations over Time after Transtympanic and Systemic Injections

Objective

Systemic steroid injections are used to treat idiopathic sudden-onset sensorineural hearing loss (ISSHL) and some inner ear disorders. Recent studies show that transtympanic (TT) steroid injections are effective for treating ISSHL. As in vivo monitoring of drug delivery dynamics for inner ear is lacking, its time course and dispersion of drugs is unknown. Here, we used a new in vivo imaging system to monitor drug delivery in live mice and to compare drug concentrations over time after TT and systemic injections.

Methods

Luciferin delivered into the inner ears of GFAP-Luc transgenic mice reacted with luciferase in GFAP-expressing cells in the cochlear spiral ganglion, resulting in photon bioluminescence. We used the Xenogen IVIS® imaging system to measure how long photons continued to be emitted in the inner ear after TT or systemic injections of luciferin, and then compared the associated drug dynamics.

Results

The response to TT and IP injections differed significantly. Photons were detected five minutes after TT injection, peaking at ∼20 minutes. By contrast, photons were first detected 30 minutes after i.p. injection. TT and i.p. drug delivery time differed considerably. With TT injections, photons were detected earlier than with IP injections. Photon bioluminescence also disappeared sooner. Delivery time varied with TT injections.

Conclusions

We speculate that the drug might enter the Eustachian tube from the middle ear. We conclude that inner-ear drug concentration can be maintained longer if the two injection routes are combined. As the size of luciferin differs from that of therapeutics like dexamethasone, combining drugs with luciferin may advance our understanding of in vivo drug delivery dynamics in the inner ear.     

Microbe-specific C3b deposition in the horseshoe crab complement system in a C2/factor B-dependent or -independent manner

Complement C3 plays an essential role in the opsonization of pathogens in the mammalian complement system, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. To understand the molecular mechanism of C3b deposition on microbes, we characterized two types of C2/factor B homologs (designated TtC2/Bf-1 and TtC2/Bf-2) identified from the horseshoe crab Tachypleus tridentatus. Although the domain architectures of TtC2/Bf-1 and TtC2/Bf-2 were identical to those of mammalian homologs, they contained five-repeated and seven-repeated complement control protein domains at their N-terminal regions, respectively. TtC2/Bf-1 and TtC2/Bf-2 were synthesized and glycosylated in hemocytes and secreted to hemolymph plasma, which existed in a complex with C3 (TtC3), and their activation by microbes was absolutely Mg(2+)-dependent. Flow cytometric analysis revealed that TtC3b deposition was Mg(2+)-dependent on Gram-positive bacteria or fungi, but not on Gram-negative bacteria. Moreover, this analysis demonstrated that Ca(2+)-dependent lectins (C-reactive protein-1 and tachylectin-5A) were required for TtC3b deposition on Gram-positive bacteria, and that a Ca(2+)-independent lectin (Tachypleus plasma lectin-1) was definitely indispensable for TtC3b deposition on fungi. In contrast, a horseshoe crab lipopolysaccharide-sensitive protease factor C was necessary and sufficient to deposit TtC3b on Gram-negative bacteria. We conclude that plasma lectins and factor C play key roles in microbe-specific TtC3b deposition in a C2/factor B-dependent or -independent manner.     

d-Xylose Isomerase from a Marine Bacterium, Vibrio sp. Strain XY-214, and d-Xylulose Production from β-1,3-Xylan

The xylA gene from a marine bacterium, Vibrio sp. strain XY-214, encoding d-xylose isomerase (XylA) was cloned and expressed in Escherichia coli. The xylA gene consisted of 1,320-bp nucleotides encoding a protein of 439 amino acids with a predicted molecular weight of 49,264. XylA was classified into group II xylose isomerases. The native XylA was estimated to be a homotetramer with a molecular mass of 190 kDa. The purified recombinant XylA exhibited maximal activity at 60°C and pH 7.5. Its apparent K _m values for d-xylose and d-glucose were 7.93 and 187 mM, respectively. Furthermore, we carried out d-xylulose production from β-1,3-xylan, a major cell wall polysaccharide component of the killer alga Caulerpa taxifolia. The synergistic action of β-1,3-xylanase (TxyA) and β-1,3-xylosidase (XloA) from Vibrio sp. strain XY-214 enabled efficient saccharification of β-1,3-xylan to d-xylose. d-Xylose was then converted to d-xylulose by using XylA from the strain XY-214. The conversion rate of d-xylose to d-xylulose by XylA was found to be approximately 40% in the presence of 4 mM sodium tetraborate after 2 h of incubation. These results demonstrated that TxyA, XloA, and XylA from Vibrio sp. strain XY-214 are useful tools for d-xylulose production from β-1,3-xylan. Because d-xylulose can be used as a source for ethanol fermentation by yeast Saccharomyces cerevisiae, the present study will provide a basis for ethanol production from β-1,3-xylan.     

Ontogeny of gene expression of group IB phospholipase A₂ isoforms in the red sea bream, Pagrus (Chrysophrys) major

The red sea bream (Pagrus major) was previously found to express mRNAs for two group IB phospholipase A₂ (PLA₂) isoforms, DE-1 and DE-2, in the digestive organs, including the hepatopancreas, pyloric caeca, and intestine. To characterize the ontogeny of the digestive function of these PLA₂s, the present study investigated the localization and expression of DE-1 and DE-2 PLA₂ genes in red sea bream larvae/juveniles and immature adults, by in situ hybridization. In the adults, DE-1 PLA₂ mRNA was expressed in pancreatic acinar cells. By contrast, DE-2 PLA₂ mRNA was detected not only in digestive tissues, such as pancreatic acinar cells, gastric glands of the stomach, epithelial cells of the pyloric caeca, and intestinal epithelial cells, but also in non-digestive ones, including cardiac and lateral muscle fibers and the cytoplasm of the oocytes. In the larvae, both DE-1 and DE-2 PLA₂ mRNAs first appeared in pancreatic tissues at 3 days post-hatching (dph) and in intestinal tissue at 1 dph, and expression levels for both gradually increased after this point. In the juvenile stage at 32 dph, DE-1 PLA₂ mRNA was highly expressed in pancreatic tissue, and DE-2 PLA₂ mRNA was detected in almost all digestive tissues, including pancreatic tissue, gastric glands, pyloric caeca, and intestine, including the myomere of the lateral muscles. In conclusion, both DE-1 and DE-2 PLA₂ mRNAs are already expressed in the digestive organs of red sea bream larvae before first feeding, and larvae will synthesize both DE-1 and DE-2 PLA₂ proteins.     

Foraging habitats and floral resource use by colonies of long- and short-tongued bumble bee species in an agricultural landscape with kabocha squash fields

Bumble bees pollinate and forage on flowers of crop and wild plants in agricultural landscapes. These interactions may depend on landscape patterns and bumble bee traits. We studied the abundance, colony density, and foraging range in long-tongued Bombus diversus Smith and short-tongued B. hypocrita Pérez, and evaluated their visits to flowers of wild plants and cultivated kabocha squash (Cucurbita maxima Duchesne). In forests in a farmland, B. hypocrita workers were trapped more frequently in the canopy. Full-sibs determined by nuclear microsatellite genotypes among workers collected in the farmland showed higher colony density and a larger foraging radius in B. hypocrita (30.8 km^?2 and 848 m) than in B. diversus (8.3 km^?2 and 723 m), respectively. Regarding wild plants, workers more frequently visited shallow flowers in B. hypocrita and deep flowers in B. diversus. These results suggest that bumble bees with different traits forage on different wild flowers in different habitats. Squash flowers were visited by both bumble bee species at similar frequency in the latter period of colony growth when males and new queens appeared. Composition of full-sib workers visiting squash and wild flowers did not depend on the number of collected workers of individual colonies, indicating that foraging on squash flowers was not associated with colony growth. Thus, growth and reproduction of bumble bee colonies may be supported by various wild plants and cultivated squash, respectively.