Assessment of poliovirus eradication in Japan: genomic analysis of polioviruses isolated from river water and sewage in toyama prefecture.

Seventy-eight poliovirus strains isolated from river water and sewage in Toyama Prefecture, Japan, during 1993 to 1995 were characterized by the PCR-restriction fragment length polymorphism (RFLP) method and by partially sequencing the VP3 and VP1 regions of the viral genome. Of these isolates, 36 were identified as Sabin vaccine strains, and 42 were identified as vaccine variant strains that had less than 1.4% nucleotide divergence from the Sabin strains, including 7 isolates with patterns different from those of Sabin strains as determined by PCR-RFLP analysis. These findings suggest that wild-type poliovirus was not circulating in Toyama Prefecture.

Synthesis of carbocyclic nucleosides bearing a cyclopropane ring.

A carbocyclic cyclopropane fused nucleoside, 9-(c-4-hydroxymethylbicyclo[3.1.0]hex-r-2-yl)-9H-adenine, has been efficiently synthesized from 2-azabicyclo-[2.2.1]hex-5-en-3-one (ABH) in 6 steps, namely cyclopropanation, -reductive amide cleavage (RAC) reaction and adenine ring construction.

Molecular phylogeny of zooxanthellate bivalves.

The aim of this research was to analyze the phylogenetic relationships of zooxanthellate bivalves belonging to the genera Tridacna, Hippopus, Fragum, and Corculum as well as to the closely related azooxanthellate bivalves belonging to Vasticardium and Fulvia. The small-subunit ribosomal RNA genes (18S rDNAs) from these bivalves were amplified by polymerase chain reaction with universal eukaryotic primers and were then sequenced. The sequence data from each species were analyzed by the neighbor-joining, maximum parsimony, and maximum likelihood methods, and phylogenetic trees were constructed. The results were essentially consistent with the morphological taxonomy of these bivalves. Thus, the zooxanthellate clams branch into two lineages, one composed of the genera Fragum and Corculum in the family Cardiidae, and the other composed of the genera Tridacna and Hippopus in the family Tridacnidae. However, present results indicate that the azooxanthellate clams analyzed (Vasticardium flavum and Fulvia mutica) are more likely to form a clade with the species of Tridacna and Hippopus than with those of Fragum and Corculum. This topology suggests that either the symbiosis with zooxanthellae occurred independently in each of two lineages, Tridacna-Hippopus and Corculum-Fragum, or the symbiosis was established in clams ancestral to the lineages of both the zooxanthellate clams and the azooxanthellate clams Vasticardium and Fulvia, and the latter lost the symbiotic relationship after the symbiotic clam lineages had diverged.

Purification and characterization of thin pili of IncI1 plasmids ColIb-P9 and R64: formation of PilV-specific cell aggregates by type IV pili.

Thin pili of the closely related IncI1 plasmids ColIb-P9 and R64 are required only for liquid mating and belong to the type IV family of pili. They were sedimented by ultracentrifugation from culture medium in which Escherichia coli cells harboring ColIb-P9- or R64-derived plasmids had been grown, and then the pili were purified by CsCl density gradient centrifugation. In negatively stained thin pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope. Gel electrophoretic analysis of purified ColIb-P9 thin pili indicated that thin pili consist of two kinds of proteins, pilin and the PilV protein. Pilin was demonstrated to be the product of the pilS gene. Pilin was first synthesized as a 22-kDa prepilin from the pilS gene and subsequently processed to a 19-kDa protein by the function of the pilU product. The N-terminal amino group of the processed protein was shown to be modified. The C-terminal segments of the pilV products vary among six or seven different types, as a result of shufflon DNA rearrangements of the pilV gene. These PilV proteins were revealed to comprise a minor component of thin pili. Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was demonstrated and may play an important role in liquid mating.

Proteins of morula-like cells in hemolymph of the giant clam, Tridacna derasa.

The morula-like cell, a hemocyte packed with many large (about 3 microns in diameter) electron-dense granules, is found only in the hemolymph of giant clams belonging to the Tridacnidae. To clarify the function of the morula-like cell, we investigated its proteins, especially those found in the large granules. Proteins with molecular weights of 64 kDa, 17 kDa and 7.4 kDa were found to be specific to this type of hemocyte. N-terminal amino acid sequence analysis revealed that the 17-kDa and 7.4-kDa proteins were novel proteins rich in aromatic amino acids. Rabbit polyclonal antibody against a synthetic peptide of the 7.4-kDa protein reacted not only with that protein but also with a larger molecular weight (about 16-kDa) protein in the morula-like cell. Examination of the N-terminal amino acid sequences showed that the 16-kDa protein is distinct from the 17-kDa protein, and Western blot analysis suggested that it is a precursor of the 7.4-kDa protein. The zooxanthellate portion of clam mantle and kidney contained proteins immunoreactive to the antibody, but the azooxanthellate portion of the mantle did not contain any immunoreactive protein. These results suggest that the morula-like cells interact with the zooxanthellae.

Immunohistochemical observations of antigen presenting cells and follicular dendritic cells in the tonsil.

In the present study, in order to investigate the behavior in the lymphatic system of phagocytic cells which incorporate potential antigen in the tonsile crypt, the distribution of HRP stained macrophages in the tonsillar system was observed with regard to changes with time. Furthermore, in the tonsillar tissue, in order to investigate the relationship among HRP marked macrophages, follicular dendritic cells and adhesion molecules, such as ICAM-1, immunological double staining was performed. It is open to discussion whether ICAM-1 plays an important role as an adhesive molecule in this migration of macrophages. Immunohistochemical double staining method detected a relation between anti-ICAM-1 antibody positive cells and antigen stained macrophages. In the stained area between anti-ICAM-1 antibodies and anti-DRC-1 antibodies, positive cells were clearly observed to have reticular form, particularly in the mature germinal center. This phenomenon implies that dendritic cells express ICAM-1 on the cell surface to contact other immunorelated cells and/or substances. In the inter-follicular space, anti-ICAM-1 antibody positive cells were observed to form a cluster composed of ICAM-1 positive cells, lymphocytes and HRP stained macrophages. This cluster may be considered the most primary stage of follicles or as triggering the creation of an inmature follicle.

Pleomorphic carcinoma of the pancreas with osteoclast-like giant cells.

We studied two cases of pleomorphic carcinoma of the pancreas with osteoclast-like giant cells. The cut surface of both tumors was firm and whitish-tan in color, with extensive hemorrhage and necrosis. Microscopically, these tumors were composed mainly of pleomorphic mononuclear cells and numerous bizarre giant cells, with a spindle-cell sarcomatoid appearance and adenocarcinomatous elements exhibiting varying degrees of differentiation. Multinucleated giant cells resembling osteoclasts were frequently located around sites of necrotic hemorrhage. Immunohistochemically, most tumor cells of sarcomatous areas and some anaplastic giant cells were positive for vimentin and cytokeratin. Both carcinoembryonic antigen and CA 19-9 were detected in tumor cells in one case, forming ducts or glands. These findings suggest that the tumors in these two cases originated from pancreatic-duct cells with mesenchymal differentiation. In contrast, osteoclast-like giant cells in both cases showed strong immunoreactivity with vimentin and with KP1 and PG-M1 (CD68), which are monoclonal antibodies that react with a histiocyte-macrophage-associated antigen; however, there was no reaction with any epithelial markers. Thus, osteoclast-like giant cells are not epithelial in nature, suggesting that their origin of histiocyte-macrophage lineage is possibly induced as a paraneoplastic product.

Ecological studies on reovirus pollution of rivers in Toyama Prefecture. II. Molecular epidemiological study of reoviruses isolated from river water.

In order to clarify the source of reovirus pollution in river water, comparative surveys have been carried out between reovirus isolates from river water and those from sewage, human or animal, by making use of the analysis of genomic RNA-migration pattern of reovirus in polyacrylamide gel electrophoresis (electropherotype). The strains of reovirus serotype 1 and 2 isolated from river water were classified into 3 and 9 electropherotypes, respectively, and 8 out of these 12 types were also found among strains isolated from sewage or human. When the monthly distribution of the river isolates classified by electropherotypes was compared with that of the sewage isolates, there were cases in which strains of the same electropherotype were simultaneously isolated from both sources. The electropherotypes of 3 isolates from pig and field rodents were different from those of the other isolates. The electropherotype of an oyster isolate coincided with that of some of the isolates from humans and river water. These results indicate that the major source of reoviruses polluting river water may be the human excretion.

Seroepidemiology of spotted fever group rickettsiae in small field rodents in Japan.

To clarify the geographic distribution of spotted fever group (SFG) rickettsiae in Japan, small field rodents captured in endemic and nonendemic areas were screened for anti-SFG rickettsia antibodies by the immunofluorescence test. Among total 716 specimens tested, 73-75% of rodents were antibody-positive against Rickettsia japonica and/or Rickettsia montana, showing different degree of antibody-positive proportions among each species of the rodents. Interestingly, these profiles were not different in the rodents from each endemic and nonendemic areas, indicating that the SFG rickettsiae are prevailing in the wider areas where patients have not been found yet.

Studies on aldose reductase inhibitors from medicinal plant of "sinfito," Potentilla candicans, and further synthesis of their related compounds.

For several years we have screened natural products having aldose reductase (AR) inhibitory activity. 3,3',4-Tri-O-methylellagic acid 4'-sulfate potassium salt (2) was isolated from a Mexican herb "Sinfito" (Potentilla candicans) as a potent AR inhibitory active constituent. 2 was more potent (IC50 = 8.0 x 10(-8)M) than ellagic acid, which is one of the natural inhibitors of AR. So we examined the synthesis of ellagic acid derivatives and found that the sulfate group is one of the important function.

Diabetes insipidus caused by lymphocytic infundibuloneurohypophysitis.

Pathologic examination at autopsy of a 74-year-old man with central diabetes insipidus revealed that he had a chronic lymphocytic inflammation limited to the infundibulum, stalk, and posterior lobe of the pituitary gland. No meningitis, sarcoidosis, or granulomas were detected, though there was evidence of chronic pancreatitis. In addition, neuronal loss with gliosis was observed bilaterally in the supraoptic and paraventricular nuclei. The unusual localized inflammatory brain lesion was considered to be responsible for the diabetes insipidus.

Ecological studies on reovirus pollution of rivers in Toyama Prefecture.

Reoviruses (reos) were isolated from river water in various areas of Toyama Prefecture. The frequency of reo isolation was higher in the river water, the basin of which has a larger human population. The degree of river contamination with reo paralleled that with the Escherichia coli group of organisms, and reos were frequently isolated from sewage, too. The high antibody-positive (greater than or equal to 1:8 or greater than or equal to 1:10) rates against reos in humans and other animals tested (swine, cattle, and field rodents) indicated their wide-spread infection with reos. These results suggested that the major source of reos present in the river water may be the excretion by humans and other animals, especially the former. Survival experiments in which reos were added into the filtered or centrifuged river water and kept at various temperatures, revealed that reos survived for more than 3 years at 5 C. In the field experiment where reos suspended in cellophane tubes were kept in an agricultural water stream in winter (water temperatures below 10 C), they survived for 6 months until the water temperature rose above 20 C in summer.

Epidemiological studies on the background of the endemic occurrence of tsutsugamushi disease in Toyama Prefecture. II. Anti-Rickettsia tsutsugamushi antibody-positive rate in inhabitants of endemic and nonendemic areas.

With a view to clarifying the actual state of inapparent infection of tsutsugamushi diseases, inhibitants of endemic and nonendemic areas were screened for anti-Rickettsia tsutsugamushi antibody (anti-Rt antibody) by the indirect immunofluorescence test. The anti-Rt antibody-positive rate in the inhabitants of the endemic area (about 50%) was statistically significantly higher than that in the nonendemic area (14.7%). The antibody titer in the inhabitants of the endemic area was 10-160, and the number of inhibitants showing a high antibody titer was 2-4 times larger than that of the nonendemic area. A total of 257 volunteers in the endemic area were analyzed for the changes in anti-Rt antibody titer over 1.5-2 years on an individual basis. An increase in the antibody titer was found in 20 inhabitants. There was no difference in the anti-Rt antibody-positive rate between male and female in either the endemic or the nonendemic area. The positive rate was also compared as to the distribution by 10 years of age. In the endemic area, there were no significant differences in the positive rate between any pair of 10-year age groups from 30s to 60s, whereas in the nonendemic area, the positive rate in the teen-age group was significantly lower than those in the age groups of 20 years or older. In Yamada district, the numbers of serum samples obtained from each age group were about the same, and the distribution of the positive rates showed a normal distribution. The nurse students having their homes in Toyama Prefecture were plotted on the map as for their anti-Rt antibody and geographical distribution. The results showed that many of them having homes in the endemic area were positive for the antibody, while some antibody-positives were scattered all over Toyama Prefecture.

Epidemiological studies on the background of the endemic occurrence of tsutsugamushi disease in Toyama Prefecture. I. Epidemiology of infection with Rickettsia tsutsugamuchi among field rodents in endemic and nonendemic areas.

In order to clarify the epidemiological background of the endemic occurrence of tsutsugamushi disease in Toyama Prefecture, Japan, since 1978, comparative surveys have been carried out between endemic and nonendemic areas. Rickettsia tsutsugamushi (Rt) was isolated at a rate of about 36% (158/439) from field rodents in the endemic area while it was not isolated from any of 280 in nonendemic areas. In all of six stations in the endemic area, a significantly high proportion of rodents were found to be Rt carriers. However, no Rt was isolated from rodents captured from July to September. The organism was isolated from rodents captured in the other months, especially in a high proportion in November when infestation of rodents with Leptotrombidium pallidum was at its peak. When the rodents were examined by indirect immunofluorescence staining, the rate of anti-Rt antibody-positive animals was about 55% (157/287) and about 17% (62/368) in endemic and nonendemic areas, respectively. Larvae of mites collected from the rodents were found to belong to four genera and 11 species. Among them L. pallidum was the only mite that had been known to be a vector of Rt. L. pallidum was found most frequently and in abundance from rodents in the endemic area, whereas it was present in very small numbers in rodents in nonendemic areas. The infestation of rodents with L. pallidum showed a seasonal variation, i.e. two peaks per year, in spring and autumn, and the number of mites detected was markedly greater in November than in spring. Rt was isolated from L. pallidum on rodents captured in the endemic area.

Primary prostatic carcinoid tumor with intracytoplasmic prostatic acid phosphatase and prostate-specific antigen.

A case of prostatic carcinoid tumor with lymph node metastases is reported. The patient was a 78-year-old male who died in ventricular fibrillation. At autopsy, a 2 X 2 cm, white, irregular tumor was found in the prostate and there were several enlarged para-aortic lymph nodes. Both specimens contained a characteristic carcinoid tumor. Argyrophil stains revealed strong positivity in the primary as well as in the metastatic tumors. Electron micrographs prepared from formalin-fixed tissue demonstrated numerous membrane-bound dense-core granules. Immunoperoxidase-labeled antibodies against both prostatic acid phosphatase and prostate-specific antigen localized in the tumor cells. The ultrastructural and immunohistochemical results support differentiation of the tumor cells toward both prostatic epithelial cells and endocrine cells. We believe that this is the first reported case of a prostatic carcinoid tumor in which specific prostatic tissue markers have been demonstrated in the tumor cells.