Purified Culture Systems for Bovine Oviductal Stromal Cells

Isolated stromal cells from the ampullary and isthmic parts of bovine oviductal tissues were cultured in monolayer and spheroid (cell aggregate) systems. Prostaglandin F2α (PGF) plays a crucial role in oviductal contraction and is produced by oviductal epithelial cells in cattle. Since stromal cells of many organs produce PGF, PGF production by bovine oviductal stromal cells was investigated. After PGF synthesis was confirmed, the utility of isolation and culture methods for oviductal stromal cells was evaluated by PGF production in the present study. The homogeneity of the cells was > 99%. PGF production of the cells was increased by tumor necrosis factor-α. The stromal cells aggregated and formed a spheroid by the treatments with several reagents. PGF production was higher in the spheroid culture than in the monolayer culture. The isolation and culture methods described here will facilitate studies of the physiological function of bovine oviductal stromal cells.     

Characterization of an Alginate Lyase,FlAlyA, from Flavobacterium sp. Strain UMI-01 and Its Expression in Escherichia coli

A major alginate lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the polysaccharide-lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I—Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.     

Factors that limit maintenance of recombinant rumen bacterium in sheep rumen

Factors limiting the maintenance of recombinant ruminal bacterium in the rumen were evaluated in vitro , using batch culture prepared from rumen fluid of sheep. Butyrivibrio fibrisolvens expressing a foreign xylanase gene ( B. fibrisolvens NO4) was used as a tested recombinant that was selectable on an erythromycin-containing agar medium. The recombinant tended to reduce its level slowly in the rumen fluid of sheep on a high hay diet, while its initial decrease was more apparent in the rumen fluid of sheep on a high concentrate diet. Incubation with cell-free ruminal fluid revealed a significant decrease of inoculated recombinant, suggesting the presence of antibacterial factors limiting maintenance of the recombinant. In particular, during the first 12 h of incubation this inhibition was more notable in culture prepared from rumen fluid of sheep given the high concentrate diet. Autoclaving the cell-free rumen fluid inactivated the inhibition. Numbers of the recombinant for inoculation did not influence the final level of survived recombinant, that is, the initial depression was larger as more recombinant was inoculated. Subculturing with xylan before inoculation and/or direct addition of xylan to the batch culture did not improve survival of the recombinant. From these results it is suggested that the level of survived recombinant is limited to 10^2–4/mL of in vitro batch culture with restricted energy supply and that initial depression of the recombinant is mainly caused by the heat-sensitive antibacterial factors not associating with microbial cells in the rumen.     

Characterization and genetic mapping of eceriferum-ym (cer-ym), a cutin deficient barley mutant with impaired leaf water retention capacity

The cuticle covers the aerial parts of land plants, where it serves many important functions, including water retention. Here, a recessive cuticle mutant, eceriferum-ym (cer-ym), of Hordeum vulgare L. (barley) showed abnormally glossy spikes, sheaths, and leaves. The cer-ym mutant plant detached from its root system was hypersensitive to desiccation treatment compared with wild type plants, and detached leaves of mutant lost 41.8% of their initial weight after 1 h of dehydration under laboratory conditions, while that of the wild type plants lost only 7.1%. Stomata function was not affected by the mutation, but the mutant leaves showed increased cuticular permeability to water, suggesting a defective leaf cuticle, which was confirmed by toluidine blue staining. The mutant leaves showed a substantial reduction in the amounts of the major cutin monomers and a slight increase in the main wax component, suggesting that the enhanced cuticle permeability was a consequence of cutin deficiency. cer-ym was mapped within a 0.8 cM interval between EST marker {"type":"entrez-nucleotide","attrs":{"text":"AK370363","term_id":"326489160","term_text":"AK370363"}}AK370363 and {"type":"entrez-nucleotide","attrs":{"text":"AK251484","term_id":"151420132","term_text":"AK251484"}}AK251484, a pericentromeric region on chromosome 4H. The results indicate that the desiccation sensitivity of cer-ym is caused by a defect in leaf cutin, and that cer-ym is located in a chromosome 4H pericentromeric region.