Seigo Kishikawa

Molecular detection of kobuvirus sequences in stool samples collected from healthy pigs in Japan

This study reports the detection and genetic characterization of porcine kobuvirus, a new member of the genus Kobuvirus in the family Picornaviridae. Among 293 fecal specimens collected from healthy pigs in 2009, in Japan, 133 (45.4%) were positive for kobuvirus using RT-PCR screening method. Of the positive specimens, 124 were obtained from pigs < or =6 months old, while 9 samples were from pigs >6 months old. Fifty-two representative strains of kobuviruses detected in this study were randomly selected and analyzed for their phylogenetic relationships with those other kobuvirus reference strains. The phylogenetic tree confirmed that 51 strains belonged to porcine kobuvirus and formed the exclusive branch with other porcine kobuvirus reference strains. In addition, the nucleotide sequence of H023/2009/JP shared very low levels of sequence identity with those of other porcine kobuvirus strains, but showed the highest level of sequence identity with bovine kobuvirus U-1 prototype strain. Our study demonstrated clearly that, porcine kobuvirus infection was common in healthy pigs and high prevalence of this virus was found in younger age of <6 months old of porcine populations in Tokyo and Hokkaido, Japan.

Application of biotinylated oligonucleotide probes to the detection of pituitary hormone mRNA using northern blot analysis, in situ hybridization at the light- and electron-microscope levels.

SummaryThere have been many reports on radioisotopic in situ hybridization (ISH) studies for the demonstration of pituitary hormone mRNAs in normal pituitary gland and pituitary adenomas. Recent studies have revealed that non-radioisotopic ISH has several advantages over the radioisotopic method. Using ISH with biotinylated oligonucleotide probes, we have been able to localize various pituitary hormone mRNAs in paraffin wax or frozen sections of rat normal pituitary gland and human pituitary adenomas. For control studies we used ISH with sense probes, ISH without probes, pretreatment with ribonuclease, ISH with a probe for β-actin and Northern blot hybridization. Using biotinylated probes, gene transcripts of rat growth hormone and prolactin were detected by Northern blot hybridization. The same biotinylated probes were used not only for light microscope ISH but also for the electron microscopical demonstration of rat growth hormone and prolactin mRNAs on the polysomes of the rough endoplasmic reticula. It is emphasized that biotinylated oligonucleotide probes are useful for the analysis of pituitary endocrine function because they are applicable to the three hybridization methods, namely, Northern blot hybridization and ISH at the light and electron microscope levels.

Phylogenetic Analysis of Field Isolates of Feline Calcivirus (FCV) in Japan by Sequencing Part of Its Capsid Gene

The molecular epidemiology of the infectious disease caused by feline calcivirus (FCV) in Japan was investigated by analysing the phylogenetic relationship among 21 Japanese field isolates, including the F4 strain, and 30 global isolates. Parts of the capsid gene (B–F) of the isolates were amplified by RT-PCR, and the amino acid sequences were compared with those from the global isolates. Thirty-seven and 14 out of a total of 51 isolates were clustered into two distinct genogroups, I and II respectively, by UPGMA and NJ analysis. Seven of the 21 Japanese isolates (33%) fell into group I together with 30 global isolates, while the other 14 Japanese isolates (67%) belonged to group II. The bootstrap repetition analysis of groups I and II formed by the NJ method gave a value of 99.0%. The 14 latter Japanese isolates were clearly separated from the isolates in group I, and they were different from any previously known FCV, forming a new genogroup, which implies that this lineage has been confined to Japan. Comparing the amino acid sequences shared by groups I and II, the amino acid at position 377 in B region was asparagine (Asn or Asp (NH2)) in group I, while it was lysine (Lys) in all the strains in group II. Similarly, the amino acid at position 539 in the F region was alanine (Ala) or proline (Pro) in group I, while it was valine (Val) in group II; glycine (Gly) at position 557 in group I was serine (Ser) in Group II; and phenylalanine (Phe) or leucine (Leu) at position 566 in genogroup I was tyrosine (Tyr) in group II.

Overexpression and genetic abnormality of p53 in parathyroid adenomas

To study the significance of p53 abnormality in parathyroid tumors, 32 parathyroid adenomas and 22 hyperplastic glands from 14 cases of secondary hyperparathyroidism were analysed using immunohistochemistry, polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP), single‐strand conformation polymorphism (SSCP) and DNA sequencing. Immunohistochemical study revealed p53 overexpression in four parathyroid adenomas, of which two showed diffuse and one showed focal nuclear pleomorphism. Genetic analysis disclosed allelic loss in one, and a point mutation (R290H) and a polymorphism (L257 L) in another of the two other adenomas with diffuse nuclear pleomorphism. No abnormalities were discovered in the other two adenomas, although one had a R72P polymorphism in exon 4. There was no evidence of malignancy of the four tumors in either clinical or pathological terms. None of the 22 hyperplastic glands showed p53 overexpression. These results demonstrate that p53 abnormality can occur in benign parathyroid adenomas and is more prevalent in those with nuclear pleomorphism than in those without.

Properties of a calicivirus isolated from cats dying in an agitated state

In June 1993, two of five pet cats kept in Yokohama city in Japan suddenly became agitated and died. Feline calicivirus (FCV) was isolated from them. One strain (FCV-S) was isolated from the spinal cord, lung and tonsil of cat 1, another (FCV-B) from the ileum, medulla oblongata and cervical spinal cord of cat 2, and a third (FCVSAKURA) from the oral cavity of one of the three surviving cats which showed no clinical signs. These three strains were equally resistant to pH 3˙0 and serologically similar to each other, but distinct from strain F9. A genetic analysis, using a 208 base pair fragment from region E of the capsid, showed that FCV-Ari had a 70˙4 per cent nucleotide and 77˙3 per cent amino acid homology and FCV-F9 had a 68˙6 per cent nucleotide and 73˙9 per cent amino acid homology with the three strains, indicating that these two strains were genetically distinct from the three new isolates. Unvaccinated cats and cats which had been vaccinated against FCV-F9 developed watery diarrhoea but did not become agitated after the administration of FCV-S. The FCV-S strain did not induce signs of excitability after it was administered orally to specific pathogen-free cats.

Immunolocalization of Parathyroid Hormone in Human Parathyroid Glands with Special References to Microwave Antigen Retrieval.

The subcellular localization of parathyroid hormone (PTH) in the normal human parathyroid glands with particular reference to microwave antigen retrieval was investigated using peroxidase-labeled PTH antibody, immunohistochemical, and immunoelectron microscopic methods. The results revealed that PTH granules existed mainly as pro-PTH on the trans side of Golgi and in the regions adjacent to Golgi apparatus. Only a small proportion of secretory granules were stored near the plasma membrane. Microwave irradiation was essential for the immunodetection of PTH.As the irradiative time extended from 1 to 30 min, the staining intensity increased, and the subcellular preservation decreased. Microwave irradiation for 15 min (with the sections in citrate buffer) with a power output of 500 W is the most ideal for PTH antigen retrieval, as well as for subcellular preservation.

Capsid protein genetic analysis and viral spread to the spinal cord in cats experimentally infected with feline calicivirus (FCV).

We investigated primitively the molecular basis of the neural spread of a feline calcivirus isolate (FCV-S) from the spinal cord of a cat that died after manifesting excitation. Experimental infections of cats with three clones from parent virus isolate FCV-S, isolated based on plaque size, were performed, and virus recovery from the spinal cord and the nucleotide and predicted amino acid sequences of the viral capsid protein region (ORF2) were compared. In the experimental infection with the one-time cloned virus (C1L1) isolated from a large plaque, the C1L1 was recovered from the spinal cord. In contrast, seven-times cloned C6L7 (from large plaque) and five-times cloned C5S2 (isolated from small plaque) were not recovered from the spinal cord. Genetic analysis of the capsid protein gene of the three viral clones revealed that four bases were different and two amino acids were different at positions 34 (Val in C6L7 and Ala in C1L1 and C5S2) and 46 (Leu in C6L7 and Pro in C1L1 and C5S2) between C6L7 (with large plaque) and C5S2 (with small plaque). The amino acid at position 434 of C1L1 was different from those of C6L7 and C5S2 (Gly in C1L1, D (Asp) in C6L7 and C5S2). From these results, the plaque size seemed not to be related to the spread of virus to the spinal cord. Clone C1L1, which spread to the spinal cord, had a difference of one amino acid from the other two clones, which may be related to the ability to spread to the spinal cord.