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.     

Conversion of (+)-Dihydroquercetin to 3,4-cis-Leucocyanidin by a Reductase Extracted from Cell Suspension Cultures of Cryptomeria japonica

A soluble, NADPH-dependent reductase catalyzing the reductionof (+)-dihydroquercetin to 3,4-cis-leucocyanidin (5,7,3',4'-tetrahydroxyflavan-3,4-cis-diol)was demonstrated in an enzyme preparation from a cell suspensionculture of Japanese cedar (Cryptomeria japonica D. Don). TheK_m value for (+)-dihydroquercetin was 48µM. The enzyme,which was purified 26.2-fold, could also catalyze the reductionof (+)-dihydrokaempferol to 3,4-cis-leucopelargonidin (5,7,4'-trihydroxyflavan-3,4-cis-diol). The enzyme had a pH optimumof 7 and a molecular weight of 133,000. It was inhibited byCu²⁺ and iodoacetate, but not by p-chloromercuribenzoate. Duringthe growth stages of the cell suspension cultures, an increasein reductase activity proceeds an increase in procyanidin content,as might be expected. (Received November 25, 1987; Accepted April 11, 1988)     

Inhibition of S-adenosylhomocysteine hydrolase by purine nucleoside analogues

To find out potent inhibitors of S-adenosylhomocysteine hydrolase (SAHase), several deazaadenosine analogues synthesized in this laboratory and some naturally occurring nucleoside analogues were examined with SAHases from yellow lupin seeds and rabbit liver. Neplanocin A, an antibiotic, inhibited both enzymes more potently than aristeromycin which was also an antibiotic and known as one of the most potent inhibitors of SAHase. The 3-deazaadenine derivatives (2'-deoxy, arabinosyl, xylosyl) inactivated lupin SAHase as potent as 3-deazaadenosine. Whereas, inhibitory activities of 1-deazaadenosine, its derivatives, and 7-deazaadenosine (tubercidin) were very weak.     

The nucleotide sequence of proline tRNAmo5UGG from Bacillus subtilis

The nucleotide sequence of a proline tRNA from Bacillus subtilis W168 was determined to be pC-G-G-G-A-A-G-U-A-G-C-U-C-A-G- C-U-U-G-G-D-A-G-A-G-C-A-C-A-U-G-G-psi-U-mo5U-G-G-m1G-A-C-C-A-U-G-G -G-m7G-U-C-G-C-A-G-G-T-psi-C-G-A-A-U-C-C-U-G-U-C-U-U-C-C-C-G-A-C-C- AOH, by the analysis of the unlabeled preparation and by post-labeling technique. This tRNAPro contained 5-methoxyuridine (mo5U) which is specifically distributed in bacillaceae at the wobble position of the anticodon.     

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.     

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.     

Analysis of intracellular doxorubicin and its metabolites by ultra-high-performance liquid chromatography

Doxorubicin, a highly effective anticancer drug, produces severe side effect such as cardiotoxicity, which is mainly caused by its metabolite, doxorubicinol. While in vitro studies by measuring cellular concentration of doxorubicin have been reported, there have been no reports on measuring cellular concentration of the metabolites. In this report, we developed a sensitive and high-throughput method for measuring cellular concentrations of doxorubicin and its metabolites by ultra-high-performance liquid chromatography. The method achieved more than 96% recovery of doxorubicin and its metabolites from cell homogenates. Using simple separation conditions, doxorubicin and its three main metabolites, and the internal standard, were separated within 3 min. The method has a limit of quantification of 17.4 pg (32.0 fmol) injected doxorubicin. This high sensitivity enables the detection and intracellular quantification of doxorubicin and its metabolite, doxorubicinol, in cell homogenates, and its use will facilitate studies of the relationship between doxorubicin pharmacokinetics and therapeutic outcome.