Munehiro Okamoto

HEPATOCYTE GROWTH FACTOR PREVENTS RENAL FIBROSIS AND DYSFUNCTION IN A MOUSE MODEL OF CHRONIC RENAL DISEASE

Chronic renal disease (CRD) is generally thought to be incurable, except through renal transplantation, and the number of patients with CRD is on the increase. Glomerulosclerosis and tubulointerstitial fibrosis represent the morphological equivalent of end-stage CRD. In this study, we demonstrated the preventive effect of hepatocyte growth factor (HGF) on the progression of renal dysfunction and fibrosis, using a spontaneous mouse model for CRD (ICGN strain). The mice progressively developed glomerular sclerotic injury, tubular atrophy, and renal dysfunction until they were 17 wk of age. When recombinant HGF was injected into these mice during a 4-wk-period (from weeks 14-17 after birth), DNA synthesis of tubular epithelial cells was found to be 4.4-fold higher than in mice without HGF injection, thereby suggesting tubular parenchymal expansion promoted by HGF. Notably, HGF suppressed the expression of transforming growth factor-beta and of platelet-derived growth factor as well as myofibroblast formation in the affected kidney. Consequently, the onset of tubulointerstitial fibrosis was almost completely inhibited by HGF, while HGF attenuated the progression of glomerulosclerosis, both leading to preventing manifestation of renal dysfunction. From our results, supplement therapy with HGF may be taken into consideration as a novel option for prevention and treatment of CRD.

Phylogenetic relationships withinTaenia taeniaeformis variants and other taeniid cestodes inferred from the nucleotide sequence of the cytochromec oxidase subunit I gene

Nucleotide sequence variations in a region of the mitochondrial cytochromec oxidase subunit I (COI) gene (391 bp) were examined within seven species of the genusTaenia and two species of the genusEchinococcus, including ten isolates ofT. taeniaeformis and six isolates ofE. multilocularis. More than a 12% rate of nucleotide differences between taeniid species was found, allowing the species to be distinguished. InE. multilocularis, no sequence variation was observed among isolates, regardless of the host (gray red-backed vole, tundra vole, pig, Norway rat) or area (Japan, Alaska) from which each metacestode had been isolated. In contrast, six distinct sequences were detected among the tenT. taeniaeformis isolates examined. The level of nucleotide variation in the COI gene withinT. taeniaeformis isolates except for one isolate from the gray red-backed vole (TtACR), which has been proposed as a distinct strain or a different species, was about 0.3%–4.1%, whereas the COI gene sequence for TtACR differed from those of the other isolates, with levels being 9.0%–9.5%. Phylogenetic trees were then inferred from these sequence data using two different algorithms.

Geographical genetic structure within the human lung fluke, Paragonimus westermani , detected from DNA sequences

Nucleotide sequences were obtained for the second internal transcribed spacer of the ribosomal gene repeat and for part of the mitochondrial-cytochrome c oxidase subunit I gene from geographical isolates of Paragonimus westermani from Japan, China, Korea, Taiwan, the Philippines, peninsular Malaysia and Thailand. Sequences were obtained from several other species of Paragonimus for comparative purposes. Two groups were recognized within P. westermani: an NE group (China, Japan, Korea, Taiwan) which was relatively uniform and included both diploid and triploid forms, and a southern group (Malaysia, Thailand, Philippines), members of which were genetically distant from one another. According to both ITS2 and COI data, genetic distances among P. westermani isolates equalled or exceeded those between some distinct species of Paragonimus. The ITS2 sequences were conserved relative to COI sequences. Substitutions among the latter may be approaching saturation within the genus Paragonimus.

Production of recombinant SERA proteins of Plasmodium falciparum in Escherichia coli by using synthetic genes

We expressed two regions of the serine repeat antigen (SERA) protein of Plasmodium falciparum in Escherichia coli by synthesizing the genes with a changed codon usage. One of the synthetic gene sequences encodes amino acid residues 17-382 (SE47) and the other encodes amino acid residues 586-802 (SE50A). The products produced by the synthetic gene sequences in E. coli accounted for 15-30% of the total bacterial protein. Antisera against both the purified gene products prepared in rats inhibited malaria parasite growth in vitro. The anti-SE47 serum was significantly more inhibitory than the anti-SE50A serum. The described methods provide a large scale preparation of recombinant antigens for improving and producing malaria vaccine.

Phylogenetic relationships of three hymenolepidid species inferred from nuclear ribosomal and mitochondrial DNA sequences

Three hymenolepidid tapeworms, Hymenolepis diminuta, H. nana and H. microstoma, are commonly maintained in laboratory rodents and used in many experimental model systems of tapeworm infections. We examined partial sequences from the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene and nuclear ribosomal internal transcribed spacer 2 (ITS2) sequences to infer phylogenetic relationships of the 3 hymenolepidid species. Parts of the CO1 gene and ITS2 were amplified by PCR and sequenced directly. The CO1 gene sequence obtained was the same in length (391 bp) among all specimens. In the case of ITS2, however, several insertions and deletions were detected (671-741 bp) not only among species but also between an American isolate and a Japanese isolate of H. diminuta. Percentage nucleotide differences between H. diminuta and H. microstoma, or H. diminuta and H. nana were 16.6-18.2% for the CO1 gene and 21.3-22.9% for ITS2. The differences in both sequences between H. microstoma and H. nana were about 14%. Phylogenetic trees inferred from both of the nucleotide sequences showed similar topology, and suggest that H. diminuta may have diverged from the common ancestral line the earliest, and that H. nana is closer to H. microstoma than to H. diminuta.

Phylogenetic identification of Sparganum proliferum as a pseudophyllidean cestode by the sequence analyses on mitochondrial COI and nuclear sdhB genes.

Sparganum proliferum is a larval cestode for which the adult stage is unknown. It is characterized by the continuous branching and budding when parasitized to humans, and causes fatal human sparganosis. However, the biological features of S. proliferum, including its taxonomic status, still remain obscure. Our previous investigation suggested that S. proliferum might be phylogenetically distinct from Spirometra erinaceieuropaei, by the analysis on mitochondrial NADH dehydrogenase subunit 3 (ND3) gene. However, mitochondrial DNA sequence in Platyhelminth is known to have heteroplasmy within a species. Therefore, in the present study, we have investigated the complete nucleotide sequences of mitochondrial cytochrome c oxidase subunit I (COI) gene and the partial nucleotide sequences of nuclear coded succinate dehydrogenase iron-sulfur protein subunit gene (sdhB). The results clearly demonstrated that S. proliferum is a distinct species from S. erinaceieuropaei, and that S. proliferum belongs to the order Pseudophyllidea.

Human Taenia eggs develop into cysticerci in scid mice

The intermediate hosts for Taenia saginata and T. solium are cattle and pigs (and humans for the latter), respectively. In vitro-hatched (but not activated) oncospheres of both Asian Taenia (T. saginata asiatica, a new subspecies of T. saginata or T. asiatica, a new species) and T. solium injected subcutaneously into the backs of mice with severe combined immunodeficiency (scid) developed into fully matured cysticerci. Five-month-old metacestodes of Asian Taenia had no hooklets and were bigger in size than those previously reported and similar to those of T. saginata. Their morphology suggested that the cysticerci were more advanced than those in the intermediate host animals. It is suggested that scid mice are valuable experimental animal models for studying human taeniid cestode infections.

Nephrotic mice (ICGN strain) : A model of diffuse mesangial sclerosis in infantile nephrotic syndrome

The ICGN mouse strain is a unique model for naturally occurring nephrotic syndrome. In the present study, we examined the onset of the clinical manifestation of nephrotic syndrome and determined the sequence of intraglomerular events associated with progression of nephrotic conditions. Laboratory analysis revealed that homozygous (nep/nep) mice showed urinary albumin excretion during the suckling stage, rapidly leading to hypoalbuminemia accompanied by body growth failure. Renal pathology demonstrated that an initial intraglomerular event in the nephrotic mice was observed 3 weeks after birth in the form of mesangiolytic lesions, characterized by microaneurysm, platelet accumulation and capillary ballooning. In 6-week-old homozygous mice, mesangial sclerosis, characterized by mesangial expansion and glomerular hypertrophy, was observed in a diffuse fashion. Immunohistochemistry revealed that the glomerular cells in the 3-week-old homozygous suckling mice were positive for α-smooth muscle actin, suggesting a phenotypic change in the mesangial cells. Mesangial expansion, confirmed by the over-deposition of type I collagen, was evident until 6 weeks after weaning, while it was of interest that fibrogenic cytokines such as platelet-derived growth factor and transforming growth factor-β were not detected in the sclerotic glomeruli throughout the observations. Furthermore, the nephrotic features were shown to be resistant to steroid therapy with a high dose of prednisolone. Our results suggest that diffuse mesangial sclerosis, a hereditary glomerular disease, may be genetically generated through early myofibroblast formation, which occurs and develops probably independently of up-regulation of these fibrogenic cytokines. In conclusion, the homozygous nephrotic mouse (ICGN strain) is believed to be a good model for investigating not only nephrotic conditions but also cellular and molecular pathogenesis of diffuse mesangial sclerosis in steroid-resistant infantile nephrotic syndrome.

Evolution of the carabid ground beetles

The phylogenetic relationships of the carabid ground beetles have been estimated by analysing a large part of the ND5 gene sequences of more than 1,000 specimens consisting of the representative species and geographic races covering most of the genera and subgenera known in the world. From the phylogenetic analyses in conjunction with the mtDNA-based dating, a scenario of the establishment of the present habitats of the respective Japanese carabids has been constructed. The carabid diversification took place ca. 40 MYA as an explosive radiation of the major genera. During evolution, occasional small or single bangs also took place, sometimes accompanied by parallel morphological evolution in phylogenetically remote as well as close lineages. The existence of silent periods, in which few morphological changes took place, has been recognized during evolution. Thus, the carabid evolution is discontinuous, alternatively having a phase of rapid morphological change and a silent phase.

Intraspecific variation ofTaenia taeniaeformis as determined by various criteria

The intraspecific variation of four laboratory-reared isolates ofTaenia taeniaformis the SRN and KRN isolates from Norwa rats,Rattus norvegicus, captured in Japan and malaysia, respectively; the BMM isolate from a house mouse,Mus musculus, captured in Belgium; and the ACR isolate from a gray red-backed vole,Clethrionomys rufocanus bedfordiae, captured in Japan was examined by various criteria. Eggs of each of the four isolates were orally inoculated into several species of intermediate host. They were most infective to the rodent species from which the original metacestode of each isolate had been isolated in the field, and only the ACR isolate was infective to the gray red-backed vole. Although little difference was found between the SRN, KRN, and BMM isolates by the other criteria, including the morphology of rostellar hooks, the protein composition of the metacestode, and restriction endonuclease analysis of DNA, the ACR isolate was clearly different from the others. It was considered that the ACR isolate was independent as a strain distinct from the other three isolates.