Development of sequence tagged site markers to identify races of Fusarium oxysporum f. sp. lactucae

By random amplified polymorphic DNA (RAPD) analysis of the representative isolates of each race of Fusarium oxysporum f. sp. lactucae, RAPD fragments of 0.6, 1.6, and 2.9kb were obtained. The 0.6-kb RAPD fragment was common to the representative isolates of all three races. Amplification of the 1.6- and 2.9-kb fragments were unique to the isolates of races 1 and 2, respectively. Sequence tagged site (STS) marker FLA0001, FLA0101, and FLA0201 were generated from the 0.6-, 1.6-, and 2.9-kb RAPD fragments, respectively. Polymerase chain reaction (PCR) analysis showed that FLA0001 was common to all 49 isolates of F. oxysporum f. sp. lactucae. FLA0101 was specifically generated from all 23 isolates of race 1 but not from races 2 or 3. FLA0201 was specifically amplified from all 12 isolates of race 2 but not from races 1 or 3. In two isolates of F. oxysporum f. sp. lactucum, PCR amplified FLA0001 and FLA0101 but not FLA0201. On the other hand, these STS markers were not detected from isolates of five other formae speciales. Because these STS markers were not generated from isolates of other plant pathogenic fungi, bacteria, or plant materials examined in this study, PCR analysis combined with the three STS markers should be a useful means for rapid identification of races of F. oxysporum f. sp. lactucae.     

Stimulation of axillary shoot formation of cuttings of Hylocereus trigonus (Cactaceae) by pre-soaking in benzyladenine solution

Either before or after curing their cut surfaces for 5 days, 7 cm- and 15-cm-long decapitated Hylocereus trigonus cuttings were treated by soaking their apical or basal ends in benzyladenine (BA) solution. They were then planted and grown in a greenhouse.For the 7 cm-long cuttings, BA (25–100 mg l^?1) applied to the apical ends for 24 h increased the ratio of cuttings with sprouted buds to 64–100%, the number of sprouted buds to 1.9–3.1 and of shoots to 1.6–2.8, and the shoot length to 35–60 mm, compared to the water control which showed 13%, 1.0, 1.0 and 12.5 mm, respectively. Soaking the basal part had only a small effect.Naphthylacetic acid (NAA) applied to the basal ends of cuttings immediately after cutting increased the number of sprouted buds and shoots by inducing early rooting. The number and length of BA-induced axillary shoots in the longer cuttings was greater than those in the shorter ones.In the 15-cm-long cuttings, increasing the soaking time from 5 min to 24 h resulted in a greater promotive effect of BA on shoot formation. BA applied before curing showed the same effect as that given after curing but caused necrosis of the tissue just under the cut surface. Enlarging the area soaked in BA solution from 5 cm to 10 cm decreased the number of sprouting buds and shoots.     

N-acetylglucosaminyltransferase I activity of bovine oviduct epithelial cells: stimulation by luteinizing hormone, vascular endothelial growth factor and tumor necrosis factor alpha

N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101), which catalyzes the first step in the conversion of oligomannose to complex or hybrid N-glycans of glycoproteins, was found in media cultured with bovine oviduct epithelial cells (BOEC) obtained from non-pregnant cows during the follicular phase. Combined treatment with specific hormones increased GnT I release from BOEC. Luteinizing hormone (LH; 10 ng/ml) alone slightly, but together with 17beta-estradiol (E2; 1 ng/ml), synergistically increased GnT I activity. Vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF) alpha, which have been shown to have their highest activities in the bovine oviduct during the periovulatory period, also increased in GnT I activity. This study provides the first evidence of an increase of GnT I release from BOEC in vitro, and shows that endocrine as well as local factors such as LH, VEGF and TNFalpha increase this activity. The results suggest that GnT I activity in the bovine oviduct may contribute to the induction of glycosylation and thereby contributing to the provision of the optimal microenvironment for fertilization and early development of the embryos.     

Acceleration of follicular development by administration of vascular endothelial growth factor in cycling female rats

To address the role of follicular angiogenesis in the determination of ovulatory follicles and the effects of different vascular endothelial growth factor (VEGF) isoforms on follicular angiogenesis and development, mature female rats were treated with an angiogenic inhibitor (TNP-470), and also with VEGF 120 or 164 at different dosages (0.4, 0.8, 4.0 or 8.0 microg/kg body weight) for 3 days during the estrous cycle. Ovarian follicular angiogenesis, the population of large follicles and ovulation were examined. VEGF 120 (0.8 microg/kg) and 164 (8.0 microg/kg) treatments stimulated follicular angiogenesis in the theca interna layer, while TNP-470 treatment showed severe depression of follicular angiogenesis, and completely inhibited ovulation. After administration of VEGF 120 or 164, the number of healthy preovulatory follicles and ovulated oocytes increased significantly, concomitantly with a decrease in the number of atretic preovulatory follicles. The oocytes ovulated had normal fertilizability and developed to term with the same litter size as in the control rats. Our findings suggest that follicular angiogenesis may be a determinant of follicular development during the periovulatory phase, and that VEGF isoforms may play different important roles in regulating follicular angiogenesis.     

Growth and maturation of follicles and oocytes following xenotransplantation of porcine ovarian tissues and in vitro maturation

This is the first report to show morphological evidence of in vitro maturation of oocytes recovered from xenotransplanted antral follicles. To develop a suitable tool for studing the growth and maturation of follicles and oocytes, we xenotransplanted small pieces of ovarian cortical tissue from sows, which contained small preantral follicles (primordial, primary, and secondary follicles; less than 0.05, 0.1 and 0.3 mm in diameter, respectively), under the capsules of kidneys of adult female severe combined immunodeficient (SCID) mice for 2 and 8 weeks, and then recovered cumulus-oocyte complexes from the growing tertiary follicles in xenografted tissues. The distribution of processes from cumulus cells to oocytes and the follicular growth, development, and maturation during xenotransplantation were histochemically analyzed. Tertiary follicles, 0.5 to 3.0 mm in diameter, were obtained from grafted tissues 2 (85%: 52 follicles/61 grafted tissues) and 8 (50%: 15/30) weeks after xenotransplantation, and then oocytes, which were tightly attached to cumulus cells, were collected from each tertiary follicle and cultured to assess their quality. At 2 weeks after grafting, 17.6% of the oocytes had matured to the metaphase II stage, but no such maturation was observed 8 weeks after grafting. Thus, in the 2 weeks group, preantral follicles rapidly grew in xenotransplanted porcine ovarian tissues to the tertiary stage, and oocytes could be recovered and matured from them by in vitro culture.     

Involvement of Phosphoglucose Isomerase in Pathogenicity of Xanthomonas oryzae pv. oryzae

ABSTRACT Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, was subjected to transposon mutagenesis to generate mutants defective in pathogenicity. A novel mutant 74M913 was attenuated in virulence but retained its ability to cause the hypersensitive response in leaf blight-resistant rice and tomato. Cloning and sequence analysis revealed that the transposon in 74M913 was inserted in a gene homologous to the phosphoglucose isomerase (pgi) gene of X. axonopodis pv. citri. Growth of the mutant in a synthetic medium containing fructose or xylose as a sole carbohydrate source was much reduced, indicating the transposon disrupted pgi function. The interaction between expression of pgi and hypersensitive response and pathogenicity (hrp) genes was investigated because we had demonstrated previously that expression of hrp genes of X. oryzae pv. oryzae is induced in a synthetic medium containing xylose. However, pgi and the hrp gene (hrcU) were expressed independently. This study suggests that PGI is involved in pathogenicity of X. oryzae pv. oryzae.     

Exon skipping of exonuclease 1 in MRL/MpJ mice is caused by a nucleotide substitution of the branchpoint sequence in intron eight

In MRL/MpJ mice, there is a genetic mutation of exonuclease 1 (Exo1), in which the exon 9 is sometimes deleted. In the present study, to check the generation of the spliced exons, exon 8-intron 8-exon 9 (pCX/Ex/EIE/B and pCX/Ex/EIE/M) plasmids were temporally transfected in vitro into BALB 3T3 cells, and RT-PCR using appropriate primer pair was carried out 1 day after transfection. In these constructions, pCX/Ex/EIE/B was derived from genomic sequence of C57BL/6 mice, and pCX/Ex/EIE/M was from MRL/MpJ. A spliced band was detected in pCX/Ex/EIE/B, but was present little or very weakly in pCX/Ex/EIE/M. Next, the same spliced band was demonstrated in the pCX/Ex/EIE/M(T) plasmid, in which the branchpoint sequence (BPS) of pCX/Ex/EIE/M including the exon 9 was changed into that of pCX/Ex/EIE/B. The splicing did not occur in the dell1/B mutant, in which 1960 nucleotides of the intron 8 were deleted, whereas it was detected in the del2/B plasmid deleted 1036 nucleotides in its middle region. These results suggest that the nucleotide T to A mutation of the BPS in the intron 8 is at least a sufficient for generation of splice variants (tr-1 and tr-2 Exo1).     

Distribution of the pores of epithelial basement membrane in the rat small intestine

The distribution and diameter of the pores of epithelial basement membrane in the intestinal villi and the lymph nodules of ileal Peyer's patches were investigated in the rat small intestine by scanning electron microscopy after the removal of the overlying epithelial cells with OsO(4) maceration. In the duodenum, jejunum and ileum, the pores were mainly distributed at the upper three fourths of the villi, but were scarce around the top of the villi. The diameter of some of the pores in the upper three fourths of the villi was larger than that of those in the lower portion. The protrusion of lymphocytes and the cytoplasmic processes of macrophages were also seen at the orifices of the pores. In ileal Peyer's patches, in contrast, pores were densely distributed in the lower one third of the follicle-associated epithelium (FAE) where M cells were mainly seen. Furthermore, these pores were larger than those found in the upper two thirds. Lymphocytes or cytoplasmic processes of macrophages were frequently seen in the lower one third of FAE. These results suggest that the pores at the basement membrane correspond to the passage of the immunocompetent cells which are in contact with M cells or villous columnar epithelial cells and that the abundance of pores is a sign of aggressive interaction between the particular epithelial cells and the immunocompetent cells at the upper three fourths of intestinal villi and the lower one third of FAE in the rat small intestine.     

Glucose uptake activity in murine red blood cells infected with Babesia microti and Babesia rodhaini

The glucose uptake activity in Babesia rodhaini and B. microti - infected red blood cell (IRBC) was investigated in mice using 2-deoxy-D-glucose (2DOG) and L-glucose (L-Glc), a non-metabolizable analogue of D-glucose and non-incorporative glucose to non-infected RBC (NRBC), respectively. The uptake activities of both DOG and L-Glc were higher in IRBCs than those in NRBC. The concentration dependent uptake of 2DOG and L-Glc in both IRBC revealed a linear curve, indicating non-transporter mediated uptake. In addition, B. microti IRBC showed higher 2DOG uptake than B. rodhaini IRBC, whereas no difference was observed in L-Glc uptake. These results indicated that some new glucose uptake system, at least two systems, developed in both IRBC. The new systems were sodium independent, non-competitive to L-Glc, and sensitive to temperature. One of two systems had no kinetical difference between B. rodhaini and B. microti IRBC, however another one might have higher uptake activity in B. microti IRBC compared to that in B. rodhaini IRBC.