Involvement of mouse and porcine PLCζ-induced calcium oscillations in preimplantation development of mouse embryos.

In mammals, phospholipase Cζ (PLCζ) has the ability to trigger calcium (Ca(2+)) oscillations in oocytes, leading to oocyte activation. Although there is a species-specific difference in the PLCζ-induced Ca(2+) oscillatory pattern, whether PLCζ-induced Ca(2+) oscillations affect preimplantation embryonic development remains unclear. Here, we show that Ca(2+) oscillations in mouse PLCζ cRNA-injected oocytes stopped just before pronuclear formation, while that in porcine PLCζ cRNA-injected oocytes continued for several hours after pronuclei had been formed.

A single nucleotide polymorphism of porcine MX2 gene provides antiviral activity against vesicular stomatitis virus.

The objective was to determine if single nucleotide polymorphisms (SNPs) in porcine MX2 gene affect its antiviral potential. MX proteins are known to suppress the multiplication of several viruses, including influenza virus and vesicular stomatitis virus (VSV). In domestic animals possessing highly polymorphic genome, our previous research indicated that a specific SNP in chicken Mx gene was responsible for its antiviral function.

Both antiviral activity and intracellular localization of chicken Mx protein depend on a polymorphism at amino acid position 631.

The Mx protein is known to inhibit the multiplication of several RNA viruses. In chickens, a polymorphism at amino acid position 631 (631 aa) of Mx protein has been suggested to be involved in the antiviral ability against vesicular stomatitis virus (VSV) and influenza virus, indicating that a Ser-to-Asn substitution at 631 aa is the source of this antiviral ability. However, how the substitution at 631 aa contributes to the antiviral activity remains to be clarified.

Specific intracellular localization and antiviral property of genetic and splicing variants in bovine Mx1.

In bovine Mx1, only an amino acid substitution between Ile and Met at position 120 was detected by the nucleotide sequence and mismatched PCR-RFLP technique. The Ile variant was assumed to distribute mainly in the bovine population since the gene frequency was 79.3%.

A PCR-based protocol for the generation of a recombinant West Nile virus.

Viral reverse genetics, particularly infectious cloning, is a valuable tool with applications to many areas of viral research including the generation of vaccine candidates. However, this technology is sometimes insufficient for the construction cDNA clones as the genome sequences and/or encoding proteins of some viral agents may be toxic to the host cells used for cloning. To circumvent this problem, we developed a polymerase chain reaction (PCR)-based protocol for generating a complete West Nile virus (WNV) cDNA.

Structural and functional analysis of the bovine Mx1 promoter.

The bovine Mx1 promoter region was found to contain 4 IFN-stimulated response elements (ISREs), 7 GC boxes, 2 IL-6 responsive elements, 2 NFκB-binding sites and 2 AP-1-binding sites. Among Holstein, Charolai, and Brahman breeds, 5 nucleotide substitutions were detected in the promoter region. After the Mx1 promoter region from Holstein had been constructed with pGL-basic expression vector, the transfected cells showed promoter activity after IFN induction.

Molecular cloning and characterization of porcine Mx2 gene.

Mx, an interferon-inducible protein, is found in various vertebrates and confers resistance to several RNA viruses. At least two Mx proteins occur in vertebrates, and these proteins are key components of innate defense against viral infection. In mice and humans, the two Mx genes have different antiviral activities.

Improvement in the in vitro maturation rate of porcine oocytes vitrified at the germinal vesicle stage by treatment with a mitochondrial permeability transition inhibitor.

In this study, we examined the effects of inhibitors of mitochondrial permeability transition (MPT), caspase activity, intracellular Ca(2+) chelator and mitochondrial Ca(2+) uniporter on survival assessed by morphological observation and in vitro maturation (IVM) of porcine vitrified germinal vesicle (GV) oocytes. When vitrified GV oocytes were matured only present in the IVM medium with an MPT inhibitor, cyclosporin A (CsA), the survival and IVM rates (36.1% and 26.

A naturally occurring variant of porcine Mx1 associated with increased susceptibility to influenza virus in vitro.

Mx1 has been implicated in resistance to the influenza virus. We have now identified four alleles of the Mxl gene in domesticated breeds of pigs. Two of the alleles encode deletion variants (a 3-bp deletion in exon 13 and an 11-bp deletion in exon 14), which might be expected to interfere with Mx activity.

Molecular cloning, testicular postnatal expression, and oocyte-activating potential of porcine phospholipase Czeta.

The molecular mechanism by which sperm triggers Ca2+ oscillation, oocyte activation, and early embryonic development has not been clarified. Recently, oocyte activation has been shown to be induced by sperm-specific phospholipase Czeta (PLCzeta). The ability of PLCzeta to induce oocyte activation is highly conserved across vertebrates.

Analysis of specific factors generating 2-cell block in AKR mouse embryos.

The phenomenon of the developmental arrest at the 2-cell stage of 1-cell embryos from some mouse strains during in vitro culture is known as the 2-cell block. We investigated the specific factors involved in the 2-cell block of AKR embryos by means of a modified culture system, the production of reconstructed embryos by pronuclear exchange and a cross experiment. In a culture medium with phosphate, 94.

Examination of the Lunatic fringe and Uncx4.1 expression by whole-mount in situ hybridization in the embryo of the CKH-Jsr (jumbled spine and ribs) mouse.

The CKH-Jsr (jumbled spine and ribs) mouse was found as a spontaneous mutant with malformation of vertebrae, that is, a short trunk and kinky tail. We examined Lunatic Fringe (Lfng) and Uncx4.1 expression in the presomitic mesoderm (PSM) and somites of Jsr-mutant (CKH-Jsr/+) embryos to elucidate pathogenesis of the Jsr mutation.

Intracellular localization and antiviral property of canine Mx proteins.

Mx is an interferon (IFN)-induced protein that shows antiviral activities against RNA viruses. We examined an expression of mRNA, an intracellular localization of protein, and an antiviral property of canine Mx1 and Mx2. Both Mx1 and Mx2 mRNAs were induced in a canine kidney cell line Madin-Darby canine kidney (MDCK), stimulated with an IFN-inducer, poly(I) x poly(C) for 12 h, suggesting the presence of regulatory mechanisms consistent with Mx genes in other species.

A functional truncated form of c-kit tyrosine kinase is produced specifically in the testis of the mouse but not the rat, pig, or human.

The complete nucleotide sequence of mouse-truncated mRNA of c-kit, tr-kit, has been determined using the CD1 strain. In this study, the nucleotide sequences of tr-kit from AKR/N, C57BL/6, and ICR strains of mice were determined and found to be identical, although many silent variations were found compared with the sequence in a database for CD1. Tr-kit protein consists of 12 amino acids encoded by the 16th intron and the following 190 amino acids of c-kit.

Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos.

After in vitro maturation and fertilization of porcine oocytes, the fertilized embryos were cultured under 5 or 20% oxygen (O2) for 7 days. In embryos cultured under 5% O2 versus 20% O2, development to the blastocyst stage was higher (36.3% versus 22.

Effects of delipidation and oxygen concentration on in vitro development of porcine embryos.

The effects of delipidation and the oxygen (O(2)) concentration in the atmosphere during culture on in vitro development and H(2)O(2) content were investigated in porcine in vivo fertilized embryos and embryos after in vitro maturation and in vitro fertilization (IVM/IVF embryos). There was no significant difference in the developmental rates to the blastocyst stage between the intact and delipidated IVM/IVF embryos. However, the mean number of cells in blastocysts derived from delipidated IVM/IVF embryos (19.

Characterization of the chicken PKR: polymorphism of the gene and antiviral activity against vesicular stomatitis virus.

The double-stranded RNA-dependent protein kinase (PKR) is induced in mouse and human cells on treatment with interferon. In this study, we have cloned and determined the nucleotide sequences of chicken PKR cDNA in various chicken breeds. Chicken PKR was a 550-amino-acid protein as deduced from the cDNA open reading frame (ORF), and there were specific domains (two double-stranded RNA binding domains (DRBDs) and numerous kinase subdomains) characterized in RNA binding proteins and kinase families.

Mouse Mx2 gene: organization, mRNA expression and the role of the interferon-response promoter in its regulation.

Mx is an interferon (IFN)-inducible intracellular protein found in various vertebrates that mediates resistance against negative-strand RNA viruses. We have demonstrated previously that feral mouse strains are able to express a functional Mx2 mRNA, while that of the inbred laboratory strains was non-functional because of a single nucleotide insertion in the open reading frame, and under the detectable level. In the present study, we examined the regulation of Mx2 expression in vivo using a congenic mouse carrying Mx1 and Mx2 genes derived from feral strain SPR.

Polymorphisms and the antiviral property of porcine Mx1 protein.

We determined the cDNA sequences of the type I interferon-inducible proteins, pig Mx1 from PK(15) and LLC-PK1 cells, and compared the antiviral activities of both Mx proteins, including Mx1 polymorphisms against vesicular stomatitis virus (VSV). Mx1 cDNA derived from PK(15) cells had an 11 bp-deletion in the 3' end of the coding region, and was estimated to encode 8 amino acid substitutions and a 23 amino acid extension compared to that from LLC-PK1 cells. VSV replication was inhibited in the 3T3 cells expressing Mx1 mRNA after the cDNA was transfected.

Sequence analysis of the NRAMP1 genes from different bovine and buffalo breeds.

The natural resistance associated macrophage protein 1 (Nramp1) has been reported to confer resistance or susceptibility to Mycobacterium bovis, Salmonella typhimurium, and Leishmania donovani in the mouse, Mus musculus. A Gly and Asp substitution at position 169 of the mouse Nramp protein is invariably associated with the resistant and susceptible phenotypes, respectively. The present study aimed to detect polymorphisms in the NRAMP1 gene from different cattle and buffalo breeds.

Genetic analysis of jumbled spine and ribs (Jsr) mutation affecting the vertebral development in mice.

The jumbled spine and ribs (Jsr) mouse was derived from a spontaneous mutation. As the phenotype, a shortened trunk and kinky tail are characteristic Jsr traits. In this study, on high resolution mapping it was found that Lunatic fringe (Lfng) mapped at the same position as Jsr.

Polymorphisms and the differential antiviral activity of the chicken Mx gene.

The nucleotide sequence of chicken Mx cDNA was reported earlier using the White Leghorn breed in Germany, but it showed no enhanced resistance to viruses. In this study, the nucleotide sequences of chicken Mx cDNA were determined in many breeds. A total of 25 nucleotide substitutions, of which 14 were deduced to cause amino acid exchanges, were detected, suggesting that the chicken Mx gene is very polymorphic.