Molecular cloning and sequence analysis of feline interferon-stimulated gene 15

The interferon-stimulated gene 15 (ISG15) is induced by type I interferon (IFN). Recent studies have revealed that like ubiquitin, ISG15 is conjugated with target proteins. In this study, the feline ISG15 (FeISG15) gene was cloned from feline IFNomega (FeIFNomega)-stimulated feline kidney epithelial (CRFK) cells. According to gene sequence results, cDNA was 474bp long and encoded a protein of 157 amino acids. The putative amino acid sequences showed 62.5-72.1% identity with those of other mammalian ISG15s. Similar to human and mouse ISG15, FeISG15 included tandem ubiquitin-like domains; its homology with feline ubiquitin was 36.3-39.5%. The LRLRGG conjugating motif was located only in the carboxyl terminal ubiquitin-like domain. FeISG15 also lacked the carboxyl terminal extension after the LRLRGG motif, which is present in mouse and human ISG15. Recombinant FeISG15 protein was expressed as a His-tagged fusion protein in Escherichia coli and purified by ion-exchange chromatography followed by affinity chromatography. Monoclonal anti-FeISG15 antibodies revealed free FeISG15 and FeISG15 conjugated with target proteins in cells after IFNomega stimulation by Western blotting analysis. Furthermore, mRNA of IFNgamma was detected from peripheral blood mononuclear cells (PBMCs) after stimulation with rFeISG15 extracellularly by RT-PCR. Taken together, these results suggested that FeISG15 had ubiquitin- and cytokine-like activity, as in other species.     

Inheritance of gramine content in barley

The inheritance of gramine indole alkaloid was studied in F1 and F2 generations derived from crosses between wild ( Hordeum vulgare subsp. spontaneum) and cultivated ( H. vulgare subsp. vulgare) barley at adult stage in the field. The means of gramine content were very low in F1s and F2s in April and May. The ratio of progeny containing gramine to those without gramine (near zero or undetectable) fit a mono-genic or di-genic model in F2 generation. However, the content of gramine might be controlled by minor genes in the gramine-containing plants. The direction of dominance was toward lower gramine content. The averages of dominance ( h/d) in F1 and F2 ranged from partial to complete. Broad-sense heritabilities were high (0.70 to 0.77). The usefulness of hybrids for breeding aphid resistance and possibility increasing palatability of grazing pasture for sheep and cattle is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antimicrobial resistance of Staphylococcus pseudintermedius isolates from healthy dogs and dogs affected with pyoderma in Japan

Staphylococcus pseudintermedius strains were isolated from healthy dogs and dogs with pyoderma in 2000-2002 and 2009. All the isolates from dogs with pyoderma in 1999-2000 and from healthy dogs in 2000-2002 and 2009 were susceptible to cefalexin and/or other cephalosporins and oxacillin. However, 7.1-12.5 and 11.4% of S. pseudintermedius isolates from dogs with pyoderma in 2009 were resistant to cephalosporins and oxacillin, respectively. All S. pseudintermedius isolates from dogs with pyoderma in 1999-2000 and those from healthy dogs in 2000-2002 were susceptible to fluoroquinolones; however, 50% of the S. pseudintermedius strains isolated from dogs with pyoderma in 2009 and 30% of the S. pseudintermedius strains isolated from healthy dogs in 2009 were resistant to fluoroquinolones. Of the 21 oxacillin-resistant S. pseudintermedius (MRSP) isolates, 11 carried SCCmec type V and 10 carried hybrid SCCmec types II-III. Staphylococcus pseudintermedius strains that were resistant to only one of three fluoroquinolones had a mutation in the quinolone resistance determination region of grlA, whereas S. pseudintermedius strains that were resistant to two or more fluoroquinolones had mutations in the quinolone resistance determination regions of both grlA and gyrA.     

Identification and characterization of feline UBE1L gene

Interferon-stimulated gene 15 (ISG15) is one of the type I interferon-inducible proteins. Addition of ISG15 known as ISGylation is an ubiquitin-like posttranslational modification. Coexpression of ISG15 and ubiquitin-activating enzyme E1-like protein (UBE1L) is required to induce ISGylation. Previously, we identified feline ISG15 gene and found that the capsid protein of feline immunodeficiency virus was ISGylated in vitro by treatment with feline interferon-ω. In this study, we cloned feline UBE1L (FeUBE1L) gene to further study the mechanism of the antiviral activities induced by ISGylation. Sequencing analysis revealed that active sites of FeUBE1L were highly conserved. These data suggest that FeUBE1L has an enzymatic activity. Further, expression of FeUBE1L was induced in feline cell lines by treatment with feline interferon-ω and ovine interferon-τ.     

Cloning and expression of canine interferon-alpha genes in Escherichia coli

We cloned five new subtypes of cDNA encoding canine interferon-alpha (CaIFN-alpha) from a canine epithelial cell line. CaIFN-alphas were divided into two groups by amino acid sequences and a molecular phylogenic tree. Two subtypes of them were expressed in Escherichia coli, and IFN proteins were purified. Recombinant CaIFN-alphas were highly species-specific and showed antiviral activity against Vesicular stomatitis New Jersey virus and canine adenovirus-1 , but not against canine herpesvirus-1.     

Cloning of cDNAs encoding multiple subtypes of feline interferon-alpha from the feline epitherial cell line

Mammalian interferon (IFN)-alpha consists of a 23-amino acid signal peptide and a 166-amino acid mature protein. Feline (Fe) IFN-alpha has an extra unique molecule consisting of a 171-amino acid mature protein with a 5-amino acid insertion. We cloned eight new subtypes of cDNA encoding FeIFN- alpha from a feline epithelial cell line. Among all the FeIFN-alpha subtypes, including six that have previously been reported, the variations were found to be far less than those of IFN-alphas of other animals.     

Mechanism of self-sterility in a hermaphroditic chordate

Hermaphroditic organisms avoid inbreeding by a system of self-incompatibility (SI). A primitive chordate (ascidian) Ciona intestinalis is an example of such an organism, but the molecular mechanism underlying its SI system is not known. Here, we show that the SI system is governed by two gene loci that act cooperatively. Each locus contains a tightly linked pair of polycystin 1-related receptor (s-Themis) and fibrinogen-like ligand (v-Themis) genes, the latter of which is located in the first intron of s-Themis but transcribed in the opposite direction. These genes may encode male- and female-side self-recognition molecules. The SI system of C. intestinalis has a similar framework to that of flowering plants but utilizing different molecules.     

Confirming the authenticity of the geographical origin of Naruto dried wakame Undaria pinnatifida using stable nitrogen isotope ratios

In aquatic ecosystems, the stable nitrogen isotope ratio (δ¹⁵N) of wakame is affected by the δ¹⁵N of its nitrogen source. Thus, the δ¹⁵N of wakame is thought to differ between those grown in different aquatic ecosystems. To confirm the authenticity of the geographic origin of Naruto dried wakame (DW), we conducted δ¹⁵N analysis. The δ¹⁵N (mean ± σ) of Naruto DW model samples (n = 72) was 11.1 ± 1.9 ‰. The δ¹⁵N confidence range of Naruto DW from the model was 5.3–16.9 ‰ (mean ± 3σ). The δ¹⁵N values of the DW model of Chinese (n = 23) and South Korean origin (n = 22) were 4.2 ± 1.4 and 1.6 ± 2.3 ‰, respectively. Within the δ¹⁵N confidence range of Naruto DW, two model samples of Chinese DW and one model sample of South Korean DW were found. From those results, DW of Naruto origin was able to be distinguished from DWs of the Chinese and the South Korean.     

Insight into nucleotide and Ca<Superscript>2+</Superscript> binding to carp α-actin

ABSTRACT:   Nucleotides and Ca²⁺ binding to α-actin prepared from ordinary skeletal muscle of carp Cyprinus carpio was studied. When bound Ca²⁺ was removed with ethylenediaminetetraacetic acid, carp α-actin denatured more rapidly than chicken α-actin. Kinetic studies of the denaturation process showed that in the absence of divalent cations, the binding constants of ATP to carp and chicken actin were 5.0 × 10⁴/M and 1.2 × 10⁵/M, respectively. Competitive binding of Ca²⁺ between actin and 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N,N',N'-tetraacetic acid (Quin 2) showed that affinity of Ca²⁺ for carp actin was also lower than that for chicken actin by a factor of 1.6. These results indicated that carp actin could relatively easily denature due to the low affinities of these ligands. Enthalpy changes upon ATP binding to carp and chicken actin were −65 kJ/mol and −110 kJ/mol, respectively. Thermodynamic analyses of our results revealed that the entropy change associated with ATP binding to carp actin was significantly smaller than that to chicken actin, suggesting that structural stabilization upon ATP binding was less effective in carp actin.     

Expression of feline interferon-alpha subtypes in Esherichia coli, and their antiviral activity and animal species specificity

Two kinds of FeIFN-alpha consisting of 166 amino acids (aa) and 171 aa were expressed in Escherichia coli, and the purified proteins were tested for antiviral activity on homologous and heterologous animal cells. Crude FeIFN induced in feline cells revealed antiviral activity on both homologous and heterologous animal cells. In contrast, both types of recombinant FeIFN-alpha revealed antiviral activity only on the feline cells. All of the FeIFN-alpha subtypes showed high activity to vesicular stomatitis virus, and the three species of feline viruses belonging to different families.