In vivo expression of GFP transgene delivered via a replicating feline leukemia virus

We previously described replication-competent feline leukemia virus (FeLV) vectors with high-level and stable expression of a green fluorescent protein (GFP) or a suicide transgene in cell cultures in vitro. Considering that FeLV might potentially be used to deliver therapeutic genes in vivo, we first evaluated the expression of the GFP gene introduced in cats by the FeLV, Rickard subgroup A (FRA) construct. Eight newborn kittens were either inoculated with pFRA-GFP plasmid DNA intradermally, or challenged intraperitoneally with FRA-GFP-infected feline fibroblasts. During a 12-week observation period, five cats were shown to be progressively viremic. Quantitative PCR and RT-PCR analyses of plasma and tissue samples from these cats showed that GFP was retained in FeLV DNA or RNA to a variable degree, ranging from 0.002 to 27.890%. Tissue DNA samples were analyzed by PCR for the status of GFP and the env-transgene complex. While the proviruses carrying the GFP transgene were shown to be minor species, all tissues, however, retained the full-length GFP transgene. Despite the occurrence of predominant species with various deletions in the viral genome, approximately 1–3% of the total cell population was GFP-positive in the lymphoid tissues as visualized by laser confocal microscopy. Co-localization of immunofluorescent cells indicated that CD3-positive T cells, dendritic cells and macrophages were the major targets for GFP expression. These findings on the detectable in vivo expression of GFP for as long as a period of 3 months could be viewed positively for contemplating a therapeutic strategy for control of FeLV infection in the cats.     

The prognostic significance of minimal residual disease in the early phases of chemotherapy in dogs with high-grade B-cell lymphoma

The prognostic significance of minimal residual disease (MRD) in the early phases of chemotherapy was examined in 36 dogs with multicentric high-grade B-cell lymphoma. Sequences of immunoglobulin heavy chain (IgH) gene fragments from lymphoma cells were amplified and used to design allele-specific primers and probes for real-time PCR. The dogs were treated with a 6-month modified version of the University of Wisconsin–Madison chemotherapy protocol (UW-25) and evaluated for the MRD level at weeks 6 and 11 of UW-25.Of the 31 dogs that remained on the protocol at week 11, 14 were found to be MRD negative (<10 tumour cells/10⁵ peripheral blood mononuclear cells [PBMCs]), whereas the other 17 were MRD positive (?10 tumour cells/10⁵ PBMCs). The progression-free survival of the dogs with MRD-negative status at week 11 (median, 337 days) was significantly longer than that of the MRD-positive dogs at the same time point (median, 196 days) (P = 0.0002). These results indicate the clinical significance of MRD as a prognostic marker in the early phase of chemotherapy.     

Impact of a 5-year winter cover crop rotational system on the molecular diversity of arbuscular mycorrhizal fungi colonizing roots of subsequent soybean

The impact of winter cover crops, specifically wheat (Triticum aestivum L.), red clover (Trifolium pratense L.), and rapeseed (Brassica napus L.) or winter fallow, on community composition of arbuscular mycorrhizal fungi (AMF) in subsequent soybean roots was investigated in a 5-year field trial on andosolic soils in Japan. Soybean roots were sampled at full-flowering and analyzed for AMF communities using a partial LSU rDNA region. Phylogenetic analysis detected 22 AMF phylotypes, including eight Glomus, three Gigaspora, two Scutellospora, three Acaulospora, two Rhizophagus, and one of Funneliformis, Diversispora, Paraglomus, and an unknown glomeromycete in the roots. The 5-year rotation of different winter cover crops or winter fallow did not impact the molecular diversity of AMF communities colonizing the roots of subsequent soybean. In all of the rotations, Glomus and Gigaspora phylotypes were common to soybean roots over the 5-year period. Redundancy analysis (RDA) demonstrated that AMF communities in the roots of subsequent soybean were not significantly different among winter cover crop rotations or fallow. However, AMF communities in soybean roots were clearly influenced by rotation year suggesting that climate or other environmental factors were more important than winter cover cropping system management.     

Influence of Physicochemical Properties of Starch on Crispness of Tempura Fried Batter

The influence of the physicochemical properties of heat‐moisturetreated and untreated starches from various plant sources on the quality of tempura fried batter was examined. Batter of each starch plus wheat protein (92:8, w/w) was fried. Quality of the resulting fried batter was determined from crispness score, water evaporation, and breaking strength. The crispness score correlated with water evaporation (r = 0.95) and breaking strength (r = 0.97), indicating that water evaporation was a reliable index for evaluation of crispness of fried batter. Determination of water evaporation was easy and simple. The crispness (favorable eating texture) of tempura coating depended largely on starch origin. Among the pasting properties of starch, temperature at maximum viscosity (r = 0.77) and breakdown/maximum viscosity (% breakdown, r = ‐0.82) correlated with water evaporation, suggesting that starch resistant to gelatinization and granule disintegration produced crispy fried batter. This observation was supported by SEM. Water evaporation (r = ‐0.82) and % breakdown (r = 0.95) correlated with degree of amylose gelatinization, indicating that amylose was one of the determinants that controls crispness of fried batter by restraining disintegration of the starch granule structure.     

Molecular Structure and Some Physicochemical Properties of Buckwheat Starches

The molecular structure and pasting properties of starches from eight buckwheat cultivars were examined. Rapid viscograms showed that buckwheat starches had similar pasting properties among cultivars. The actual amylose content was 16–18%, which was lower than the apparent amylose content (26–27%), due to the high iodine affinity (IA) of amylopectin (2.21–2.48 g/100 g). Amylopectins resembled each other in average chain‐length (23–24) and chain‐length distributions. The long‐chains fraction (LC) was abundant (12–13% by weight) in all the amylopectins, which was consistent with high IA values. The amyloses were also similar among the cultivars in number‐average DP 1,020–1,380 with 3.1–4.3 chains per molecule. The molar‐based distribution indicated that all the amyloses comprised two molecular species differing in molecular size, although the weight‐based distribution showed a single species. A comparison of molecular structures of buckwheat starches to cereal starches indicated buckwheat amylopectins had a larger amount of LC, and their distributions of amylose and short chains of amylopectin on molar basis were similar to those of wheat and barley starches.