Enucleation after fusion and activation enhances the development of reconstructed bovine embryos.

Effects of enucleation timing on enucleation rates, development and methylation levels of reconstructed bovine embryos were investigated. However, the enucleation rate of reconstructed embryos produced by the enucleation before fusion and activation (EBFA) was higher than that by the enucleation after fusion and activation (EAFA) procedure (80.7% vs. 59.1%, P<0.05). The blastocyst rate of reconstructed embryos cloned with ear fibroblasts in EBFA group was reduced (P<0.05) in comparison with that of EAFA group (24.6% vs. 34.4%). Two out of 11 recipients were pregnant and gave birth to two viable calves after transfer of 20 reconstructed EBFA embryos. Two out of seven recipients were pregnant and also gave birth to two calves, with one surviving, after transfer of 12 reconstructed embryos produced by EAFA procedure. Finally, the methylation level of satellite I gene of donor cells (69.8%) and reconstructed embryos in EBFA group (64.7%) were similar, which were both higher (P<0.05) than that of the reconstructed embryos in EAFA group (44.4%). The methylation level of satellite I gene of the reconstructed embryos in the IVF embryos (31.9%) was lower (P<0.05) than those in all other treatments. In conclusion, the reconstructed bovine embryos produced by the EAFA procedure revealed a better developmental competence with a lower methylation rate of satellite I gene than those produced by the EBFA procedure.

Characterization and heterologous expression of a novel lysophospholipase gene from Antrodia cinnamomea.

AIMS: A novel lysophospholipase (LysoPL) from the basidiomycetous fungi Antrodia cinnamomea named ACLysoPL was cloned, heteroexpressed in Escherichia coli and characterized. METHODS AND RESULTS: The gene encoding ACLysoPL was obtained from expressed sequence tags from A. cinnamomea. The full length of this gene has a 945 -bp open reading frame encoding 314 amino acids with a molecular weight of 35.5 kDa. ACLysoPL contains a lipase consensus sequence (GXSXG) motif and a Ser-His-Asp catalytic triad. A putative peroxisomal targeting signal type 1 was found in the C-terminal. Heterologous expression of ACLysoPL in E. coli showed that the enzyme preferentially hydrolyses long-chain acyl esterases at pH 7 and 30 degrees C. ACLysoPL is a psychrophilic enzyme about 40% of whose maximum activity remained at 4 degrees C. The LysoPL activities with lysophospholipids as substrate were analysed by gas chromatography/mass spectrometry. CONCLUSION: We have identified and characterized a gene named ACLysoPL encoding a protein performing LysoPL and esterase activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first LysoPL of A. cinnamomea identified and characterized at the molecular level.

Essential oil from leaves of Cinnamomum osmophloeum acts as a xanthine oxidase inhibitor and reduces the serum uric acid levels in oxonate-induced mice.

The xanthine oxidase (XOD) inhibitory activity and anti-hyperuricemia effect in mice of Cinnamomum osmophloeum, which is an endemic tree in Taiwan, were evaluated in this study. The results demonstrated that the essential oil of C. osmophloeum leaves presented the strongest XOD inhibition activity (IC(50)=16.3 µg/ml); however, no significant XOD inhibition activities were found in ethanolic and hot water extracts. Furthermore, among the main compounds of essential oil, the cinnamaldehyde exhibited the potent XOD inhibition activity with an IC(50)=8.4 µg/ml. Besides, the reducing serum uric acid levels in oxonate-induced mice by cinnamaldehyde were further investigated. The hyperuricemic mice were oral administrated cinnamaldehyde at a dosage of 150 mg/kg, the uric acid value in serum was reduced from 5.25±0.63 to 2.10±0.04 mg/dl, the levels of serum uric acid in mice was lowered down by 84.48% as compared to the hyperuricemic control group. Based on the results obtained in this study, cinnamaldehyde may be a potential lead compound for developing the pharmaceutic for anti-hyperuricemia agent.

The effect of activation treatments on the development of reconstructed bovine oocytes.

The purpose of this study was to investigate the effect of different activation treatments on the development of IVM-derived and cloned bovine embryos. The effect of oocyte age (20h versus 24h after IVM) on the blastocyst rate was also investigated. No differences in the percentage of blastocyst development were observed between the oocytes matured for 20 and 24h (15% versus 27%, p>0.05). Reconstructed oocytes activated 4h after fusion (fusion before activation, FBA) had a higher blastocyst rate than those oocytes activated immediately after electrofusion (fusion and activation simultaneously, FAS) (26% versus 5%, p<0.01). Blastocyst rates were significantly greater (p<0.01) for the reconstructed oocytes activated by calcium ionophore (A23187) combined with 6-dimethylaminopurine (6-DMAP) (51.6%) than for those activated with cycloheximide (CHX) plus cytochalasin B (CB) treatment (1h, 8.2%; 5h, 14.3%). However, the blastocyst rates were similar among reconstructed oocytes activated by electric pulses and A23187 (30.5% versus 42.2%) or by A23187 and ionomycin (36.7% versus 33.3%) combined with 6-DMAP, respectively. Blastocysts that developed from reconstructed oocytes activated by A23187 and 6-DMAP resulted in three pregnancies (3/9) and one live birth from 18 embryos transferred to recipient cows. Genotypic analysis of six bovine microsatellite markers by polymerase chain reaction confirmed that the cloned calf was genetically identical to the nuclear donor. In conclusion, reconstructed oocytes that derived from oocytes exposed to activation treatment 4h after electrofusion are more likely to develop to the blastocyst stage. The best treatment to activate reconstructed bovine oocytes in this study was A23187 combined with 6-DMAP.

The effect of electrical field strength on activation and development of cloned caprine embryos.

The activation procedure used in nuclear transfer (NT) is one of the critical factors affecting the efficiency of animal cloning. The purpose of this study was to compare the effect of two electrical field strengths (EFS) for activation on the developmental competence of caprine NT embryos reconstructed from ear skin fibroblasts of adult Alpine does. The NT embryos were obtained by transfer of the quiescent fibroblasts at the fourth passage into the enucleated metaphase II (M II) oocytes. Four to five hours after electrical fusion, the NT-embryos were activated by EFS either at 1.67 or at 2.33 kV/cm and immediately incubated in 6-DMAP (2 mM) for 4 h. The cleavage rate of the NT-embryos activated with 2.33 kV/cm was greater than that activated with 1.67 kV/cm after in vitro culture for 18 h (65.6% versus 19.6%, p < 0.001). No pregnancy was found in 14 recipient does after transferring 51 NT embryos at 1-2 cell stages activated with 1.67 kV/cm. In contrast, two of the seven recipients were pregnant and gave birth to three kids after transferring 61 NT embryos at 1-2 cell stages activated by 2.33 kV/cm. The birth weights of three cloned kids were within the normal range of Alpine goats. However, one kid died 1h after birth while the remaining two are still healthy. DNA analysis by polymerase chain reaction (single-strand conformation polymorphism, SSCP) confirmed that the three kids were genetically identical to the nuclear donor.

Completion of the Genome Sequence of Watermelon silver mottle virus and Utilization of Degenerate Primers for Detecting Tospoviruses in Five Serogroups.

ABSTRACT The nucleotide sequence of the L RNA of Watermelon silver mottle virus (WSMoV) was determined. Combined with the previous work on M and S RNAs, the whole genomic sequence of this member of the genus Tospovirus was completed. The L RNA is 8,917 nucleotides in length, with one large open reading frame encoding a translation product of 2,878 amino acids (331.8 kDa) on the viral complementary strand. The L protein shares amino acid identities of only 44.3 and 46.5% with Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus, respectively; but an amino acid identity of 91.3% with Peanut bud necrosis virus. Among the sequenced tospoviruses, L protein was the most conserved gene product, whereas the nonstructural S protein was generally the most variable. Comparison of the deduced L protein of WSMoV with those of other members of the family Bunyaviridae revealed that its amino acid sequence includes the reported conserved motifs of RNA-dependent RNA polymerases. To develop a method for detecting tospo-viruses by reverse transcription-polymerase chain reaction (RT-PCR), two pairs of degenerate primers were designed from conserved regions of the L genes and used to amplify the corresponding regions of the L genes from total RNAs extracted from plant tissues infected with five serologically distinct tospoviruses. The DNA fragments obtained were identified as those of tospoviruses by restriction enzyme digestion and DNA sequencing. For field samples, watermelon and wax gourd infected with WSMoV, and lisianthus infected with TSWV were also successfully detected by these two pairs of degenerate primers, with a sensitivity similar to N-gene-specific primers. The results indicated that the RT-PCR with the degenerate primers is a fast and reliable method for detecting tospoviruses in different serogroups.

Serological and Molecular Characterization of Peanut chlorotic fan-spot virus, a New Species of the Genus Tospovirus.

ABSTRACT To clarify the serological relationship of Peanut chlorotic fan-spot virus (PCFV) with other tospoviruses, antisera were produced against the nucleocapsid (N) proteins of this virus and tospoviruses from four serogroups including Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Groundnut ringspot virus (GRSV), and Watermelon silver mottle virus (WSMoV). In immunodiffusion tests, the antisera only reacted with their homologous antigens. Similar results were noticed in indirect enzyme-linked immunosorbent assay and immunoblot tests, with the exception that strong cross-reactions were observed in heterologous combinations between TSWV and GRSV. The results indicated that the N protein of PCFV is not serologically related to those of the tospoviruses from the four serogroups. To further characterize the virus, viral S double-stranded RNA was extracted from PCFV-infected Chenopodium quinoa and used for cDNA cloning and sequencing. The full-length viral strand of the S RNA was determined to be 2,833 nucleotides, with an inverted repeat at the 5' and 3' ends and two open reading frames in an ambisense arrangement. The 3'-terminal sequence (5'-AUUGCUCU-3') of the viral S RNA is identical to those of other tospoviruses, indicating that PCFV belongs to the genus Tospovirus. The N and the NSs proteins of PCFV share low amino acid identities (22.3 to 67.5% and 19.3 to 54.2%) with those of reported tospoviruses, respectively. The phylogenetic dendrogram of the N gene of PCFV compared with those of other tospoviruses indicates that PCFV is distinct from other tospoviruses. In hybridization analyses, an N gene cDNA probe of PCFV did not react with viral RNAs of TSWV, GRSV, INSV, and WSMoV, and vice versa. Thus, based on these results, we conclude that PCFV is a new tospovirus species.

Comparison of ambisense m RNA of watermelon silver mottle virus with other tospoviruses.

ABSTRACT Double-stranded genomic RNAs (dsRNAs) extracted from Chenopodium quinoa infected with watermelon silver mottle virus (WSMV) were similar to those of tomato spotted wilt virus (TSWV, serogroup I) and impatiens necrotic spot virus (INSV, serogroup III), except that the S dsRNA of WSMV is 0.75 and 0.6 kbp longer than those of TSWV and INSV, respectively. The complete nucleotide sequence of the genomic M RNA of WSMV was determined from cDNA clones generated from separated M dsRNA. The M RNA is 4,880 nucleotides in length with two open reading frames (ORFs) in an ambisense organization. The M RNA-encoded nonstructural (NSm) ORF located on the viral strand encodes a protein of 312 amino acids (35 kDa), and the G1/G2 ORF located on the viral complementary strand encodes a protein of 1,121 amino acids (127.6 kDa). The RNA probe corresponding to the NSm or G1/G2 ORF of WSMV failed to hybridize with the M dsRNAs of TSWV and INSV. Comparison of M and S RNAs of WSMV, TSWV, INSV, and peanut bud necrosis virus (PBNV, serogroup IV) revealed a consensus sequence of eight nucleotides of 5'-AGAGCAAU...-3' at their 5' ends and 5'-...AUUGCUCU-3' at their 3' ends. The low overall nucleotide identities (56.4 to 56.9%) of the M RNA and the low amino acid identities of the NSm and G1/G2 proteins (30.5 to 40.9%) with those of TSWV and INSV indicate that WSMV belongs to the Tospovirus genus but is phylogenetically distinct from viruses in serogroups I and III. The M RNA of WSMV shares a nucleotide identity of 79.6% with that of PBNV, and the two viruses share 83.4 and 88.7% amino acid identities for their NSm and G1/G2 proteins, respectively. It is concluded that they are two related but distinct species of serogroup IV. In addition to the viral or viral complementary full-length M RNA, two putative RNA messages for the NSm gene and the G1/G2 gene, 1.0 and 3.4 kb, respectively, were detected from the total RNA extracted from WSMV-infected tissue of Nicotiana benthamiana. The 1.0- and 3.4-kb RNAs were also detected in the viral RNAs extracted from purified nucleocapsids, suggesting that the putative messages of the M RNA of WSMV can also be encapsidated by the nucleocapsid protein.