Revisiting [3 + 3] route to 1,3-cyclohexanedione frameworks: hidden aspect of thermodynamically controlled enolates.

We have revisited the traditional consecutive Michael-Claisen [3 + 3] process (MC-[3 + 3]) promising the synthesis of a cyclohexane-1,3-dione derivatives from nonactivated simple ketones and enoates and evaluated its potential in modern organic synthesis. Twenty to thirty examples were demonstrated to be effective. The reactions exhibited remarkable regioselectivity with the Michael addition proceeding through nucleophilic attack by the more hindered site of the ketones without exception. The subsequent Claisen condensation resulted in the formation of carbon-carbon bonds between less hindered site of the ketones and acyl carbon of the enoates. The MC-[3 + 3] process described is useful for the synthesis of Taxol A-ring synthons in multigram quantities and for the synthesis of other six-membered carbocyclic compounds. A number of control experiments have been conducted to provide strong support for the mechanism of this MC-[3 + 3].

Subcellular localization of Dna-initiation proteins of Bacillus subtilis: evidence that chromosome replication begins at either edge of the nucleoids.

We examined the intracellular distribution of Bacillus subtilis Dna-initiation proteins by immunofluorescence microscopy to visualize the initiation complex of replication in vivo. DnaA was distributed throughout the cytoplasm, but both DnaB and DnaI were always detected as foci during the cell-division cycle. Interaction of DnaI with the DnaC helicase by the yeast two-hybrid assay suggests that DnaI acts as a helicase loader. The number of DnaB and DnaI foci within the cell exceeded that of oriC. Although the foci were not always co-localized with oriC, they seemed to be localized near the outer or inner edges of the nucleoids at initiation of replication. When the replication cycle was synchronized in cells using a temperature-sensitive dnaA mutant, duplication of the oriC region was observed predominantly near an edge of the nucleoid. Before initiation occurred, each one of the DnaB and DnaI foci was frequently observed near there. Furthermore, DnaX-GFP (DnaX is a component of DNA polymerase III) foci were detected near either of the edges of the nucleoids at the onset of replication. These results suggest that the replisome is recruited into oriC near either edge of the nucleoids to initiate chromosome replication in B. subtilis.

Incidence of congenital rubella syndrome and influence of the rubella vaccination program for schoolgirls in Japan, 1981-1989.

In 1984, the authors first conducted a nationwide survey of deaf children with a history of maternal rubella (HMR) in special schools for the deaf in Japan. The survey showed that the incidence of congenital rubella syndrome in the Japanese mainlands was similar to those in the United States and Europe. In 1993, a second nationwide survey by the authors evaluated the efficacy of the vaccination program for schoolgirls begun in 1977. This second survey yielded 272 deaf children with HMR born between 1981 and 1989. Per 100,000 annual livebirths, the incidence rates were 1.56-9.95 in the epidemic years 1981-1982 and 1987-1988 and 0.20-0.72 in the interepidemic years. During the 1987-1988 epidemic, the incidence rates per 100,000 livebirths were 1.52 among mothers eligible for the vaccination program and 5.52-7.44 among mothers not eligible, and the difference was significant. However, only 21.7% of the women who delivered children during the 1987-1988 rubella epidemic were eligible for the vaccination program, and because the majority of deaf children with HMR were born to mothers not eligible, a decrease in the birth rate of deaf children with HMR was not observed. These data suggested the need to introduce a new vaccine program to suppress rubella epidemics.

Laxative poisoning: toxicological analysis of bisacodyl and its metabolite in urine, serum, and stool.

An 11-year-old boy was hospitalized because of severe diarrhea (over 5 L/day). The child survived; however, the diarrhea continued while he was given intravenous fluids and his electrolyte balance was closely supervised. Based on observation of the patient and his family in the hospital, surreptitious administration of a poison by his mother was suspected, and toxicological analysis was carried out on stools from the boy and on medicine administered to him by his mother. Bisacodyl, a cathartic with a direct effect on the colon, was detected in the medicine, and a metabolite of bisacodyl was present in the stool. We devised a sensitive and reliable method to quantitate the bisacodyl metabolite in urine and serum. The sample was incubated with beta-glucuronidase at 37 degrees C for 2 h; bisacodyl metabolite was extracted with tert-butyl methyl ether, then derivatized by methylation, and subjected to gas chromatography-mass spectrometry. Bisphenol A was used as an internal standard. The calibration curve was linear in the concentration range from 2 to 1000 ng/0.2 mL, and the lower limits of detection were 1 ng/0.2 mL for the urine and 2 ng/0.2 mL for the serum. As concentrations of bisacodyl metabolite in the urine and serum of the patient were clearly defined, perhaps such investigations are warranted before extensive clinical therapy is prescribed.

Mechanism of anucleate cell production in the oriC-deleted mutants of Bacillus subtilis.

We constructed oriC-deleted mutants of Bacillus subtilis by integrating the minimal replication region of plasmid pLS32 into the proA (115 degrees), spoIIIJ (360 degrees) and thrS (256 degrees) loci of the chromosome, respectively. All three mutants produced anucleate cells and the DNA/protein ratio was lower than that of the wild-type strain when grown in nutrient broth. However, when grown in minimal-glucose medium, the frequency of anucleate cells was reduced in all of them and the DNA/protein ratio was restored to normal. Especially, the oriC-deleted mutant in which the plasmid was integrated near oriC produced almost no anucleate cell. These results indicate that initiation frequency of chromosome replication from the integrated plasmid origin were reduced disproportionately to cell mass increase in rich medium, which in turn disrupted coordination between DNA replication cycle and cell division cycle. The locations of the plasmid origin relative to the natural oriC locus affected the production of anucleate cell remarkably, suggesting that partition mechanism of chromosome was also impaired by the translocation of its replication origin.

Intrapulmonary Mycobacterium avium infection as the first manifestation of chronic granulomatous disease.

A 10-month-old Japanese male infant, with no history of being prone to infections, contracted an intrapulmonary mycobacterial infection. After 2 months of intermittent fever, radiological examinations revealed mass lesions in the lung and mediastinum. Biopsy specimens showed granulomas with caseous necrosis, from which Mycobacterium avium was isolated. There was no history of mycobacteriosis or immunodeficiency diseases among his relatives. Analyses of the O2- release and expression of NADPH oxidase components verified that he suffered from gp91-phox- chronic granulomatous disease (CGD), and that his mother was a carrier of the disease. This is a rare clinical presentation for the onset of CGD, suggesting that the invasive mycobacteriosis might result from defective intracellular killing of CGD-phagocytes.