The Complete Nucleotide Sequence and Biotype Variability of Papaya leaf distortion mosaic virus

ABSTRACT The complete nucleotide sequence of the genome of Papaya leaf distortion mosaic virus (PLDMV) was determined. The viral RNA genome of strain LDM (leaf distortion mosaic) comprised 10,153 nucleotides, excluding the poly(A) tail, and contained one long open reading frame encoding a polyprotein of 3,269 amino acids (molecular weight 373,347). The polyprotein contained nine putative proteolytic cleavage sites and some motifs conserved in other potyviral polyproteins with 44 to 50% identities, indicating that PLDMV is a distinct species in the genus Potyvirus. Like the W biotype of Papaya ringspot virus (PRSV), the non-papaya-infecting biotype of PLDMV (PLDMV-C) was found in plants of the family Cucurbitaceae. The coat protein (CP) sequence of PLDMV-C in naturally infected-Trichosanthes bracteata was compared with those of three strains of the P biotype (PLDMV-P), LDM and two additional strains M (mosaic) and YM (yellow mosaic), which are biologically different from each other. The CP sequences of three strains of PLDMV-P share high identities of 95 to 97%, while they share lower identities of 88 to 89% with that of PLDMV-C. Significant changes in hydrophobicity and a deletion of two amino acids at the N-terminal region of the CP of PLDMV-C were observed. The finding of two biotypes of PLDMV implies the possibility that the papaya-infecting biotype evolved from the cucurbitaceae-infecting potyvirus, as has been previously suggested for PRSV. In addition, a similar evolutionary event acquiring infectivity to papaya may arise frequently in viruses in the family Cucurbitaceae.     

Determination of optimal conditions for parthenogenetic activation and subsequent development of rat oocytes in vitro

The present study was undertaken to determine optimal conditions for parthenogenetic activation and subsequent development of rat oocytes. Oocytes from immature Wistar-Imamichi (WI) and Sprague Dawley (SD) rats were activated by electrical stimulation in combination with 6-dimethylaminopurine (6-DMAP) to assess whether different rat strains display different responses to activation treatment. Since the cleavage rates of activated oocytes were significantly higher in WI than SD strain rats, WI rats were used for the subsequent experiments to determine the effects of post-hCG time, culture duration, different activation protocols (electrical stimulation with 6-DMAP or ionomycin with 6-DMAP) and osmolarity of the activation medium on the activation and subsequent development of WI rat oocytes. For oocytes activated by electrical stimulation combined with 6-DMAP, the percentages of oocytes that were activated and that developed to blastocysts were higher when oocytes were collected at 18-20 h than at any other time points after hCG injection (16, 22-24 h). Culturing for 2-6 h before activation treatment markedly decreased the percentage of activated oocytes that developed to beyond the four-cell stage. There were no differences in the percentages of oocytes with pronuclear formation and subsequent development to the two-cell and blastocyst stages between oocytes that were activated by electrical stimulation or ionomycin, both followed by 6-DMAP treatment. Activation of oocytes by ionomycin and 6-DMAP, both in low osmolarity media (246 mOsM), markedly increased the cleavage rates and percentages of high quality blastocysts (71%). The optimal conditions determined in the present study with simplified activation protocols and high efficiency of activation and subsequent development of WI rat oocytes will be helpful for further research involving nuclear transfer in the rat.     

Macroscopic separation of dense fluid phase and liquid phase of phosphorus

Structural transformation between a dense molecular fluid and a polymeric liquid of phosphorus that occurred at about 1 gigapascal and 1000 degrees C was investigated by in situ x-ray radiography. When the low-pressure fluid was compressed, dark and round objects appeared in the radiograph. X-ray diffraction measurements confirmed that these objects were the highpressure liquid. The drops grew and eventually filled the sample space. Decompressing caused the reverse process. The macroscopic phase separation supported the existence of a first-order phase transition between two stable disordered phases besides the liquid-gas transition. X-ray absorption measurements revealed that the change in density at the transition corresponds to about 40% of the density of the high-pressure liquid.     

Effects of water activity and lipid addition on secondary structure of zein in powder systems

The effects of water activity (A(w)) and lipid addition on the secondary structure of powdery zein were investigated using Fourier transform infrared spectroscopy. Two fatty acid esters, i.e., the linolenic and eicosapentaenoic acid ethyl esters (LAE and EPE), were mixed with the zein powder. The powders were stored in the "dry" state (with silica gel) and the "humid" state (A(w) = 0.9). The powdery zein without the lipids was shown to have a high content of the intermolecular hydrogen-bonded beta-sheet in the "dry" state, indicating the presence of protein aggregates. An increase in A(w) induced a decrease in this beta-sheet, concomitant with increases in the alpha-helix and beta-turn structures. The addition of LAE caused decreases in the alpha-helix and intermolecular hydrogen-bonded beta-sheet of zein when the powder was stored in the "humid" state, suggesting the strong interaction of LAE and zein molecules. However, LAE did not affect the secondary structure of zein in the "dry" state. The addition of EPE hardly influenced the secondary structure of zein, irrespective of A(w). These results are discussed in relation to the antioxidative activity of zein in the powder system, which had studied previously.     

Changes in qualities and quantities of consecutively ejaculated feline semen

Cats show repeated copulation, but changes in semen qualities and quantities with repetition of ejaculation have not previously been clarified. We collected semen 4 times consecutively from 5 cats using the artificial vagina method and observed the semen qualities and quantities. No significant changes were noted in the semen volume, frequency of abnormal sperm or incidence of immature sperm, but the number of sperm and sperm motility and viability decreased with repetition, and in particular, the number of sperm in the first semen accounted for 55.0% of the total number in the 4 consecutive ejaculations, showing a significant difference from those in the 2nd-4th semen (P<0.01).     

Surgical intrauterine insemination with cat semen cryopreserved with Orvus ES paste or sodium lauryl sulfate

The mean post-thaw sperm motilities of feline frozen semen prepared with 1% OEP or 3 g/ml SLS as a cryoprotective agent, in addition to 7% glycerin, were 35.0 ± 2.4 and 37.0 ± 2.5%, respectively, showing no significant difference. On unilateral intrauterine insemination (UIUI) using these semen samples at a sperm number of 40 × 10(6), the conception rate was 70.0% (7/10) in the OEP group and 30% (3/10) in the SLS group, showing that the rate was higher in the OEP group, but the difference was not significant. It was suggested that sperm in frozen semen showing the above qualities were transferred to the contralateral uterine horn on UIUI.     

The effect of ovary storage and in vitro maturation on mRNA levels in bovine oocytes; a possible impact of maternal ATP1A1 on blastocyst development in slaughterhouse-derived oocytes

Since BSE testing of slaughtered cattle is obligatory in Japan, storage of ovaries at 15-20 C overnight in phosphate buffered saline has become a routine protocol in in vitro production (IVP) of cattle embryos. Ovary storage is known to reduce developmental competence of oocytes; however, its effects on oocyte gene expression have not been clarified yet. This study compared oocytes collected from stored slaughterhouse-derived ovaries with those collected by Ovum Pick-Up (OPU) in terms of the expression of 20 selected genes to determine if ovary storage affects cellular processes at the molecular level. Expression of mRNA in oocytes was assayed before and after in vitro maturation (IVM) by real-time quantitative PCR. Maternal mRNA levels of genes were investigated in 2-cell stage embryos obtained from slaughterhouse oocytes to assess their roles for blastocyst formation. In immature OPU oocytes, genes related to metabolism (GAPDH), transporters (GLUT8, ATP1A1) and stress resistance protein (HSP70) showed significantly higher expression compared with oocytes derived from stored ovaries. During IVM, the expression of GDF9, GLUT8, CTNNB1 and PMSB1 was significantly decreased irrespective of oocyte source. Two-cell stage embryos cleaving at 22-25 h after in vitro fertilization (IVF) showed a significantly higher blastocyst formation rate and ATP1A1 gene expression level compared with those cleaving at 27-30 h after IVF. Our results reveal that storage of ovaries alters mRNA levels in oocytes. Correlation of Na/K ATPase ATP1A1 expression in IVP embryos at the 2-cell and 8-cell stages with their developmental ability to the blastocyst stage may suggest the importance of maternal mRNA of this gene during blastulation in embryos derived from slaughterhouse oocytes.