Synthesis of 4,4-Disubstituted 3,4-Dihydropyrimidin-2(1H)-ones and -thiones, the Corresponding Products of Biginelli Reaction Using Ketone, and Their Antiproliferative Effect on HL-60 Cells.

An efficient synthetic method for novel 4,4-disubstituted 3,4-dihydropyrimidin-2(1H)-ones 5 and -thiones 6 was developed. The cyclocondensation reaction of O-methylisourea hemisulfate salt 11 with 8 gives a tautomeric mixture of dihydropyrimidines 12 and 13 following acidic hydrolysis of the cyclized products to produce 5 in high yields. Thionation reaction of 5 at the 2-position smoothly proceeds to give 2-thioxo derivatives 6. These compounds 5 and 6, corresponding to the products of a Biginelli-type reaction using urea or thiourea, a ketone and a 1,3-dicarbonyl compound, have long been inaccessible and hitherto unavailable for medicinal chemistry. These methods are invaluable for the synthesis of 5 and 6, which have been inaccessible by conventional methods. Therefore, the synthetic methods established in this study will expand the molecular diversity of their related derivatives. These compounds were also assessed for their antiproliferative effect on a human promyelocytic leukemia cell line, HL-60. Treatment of 10 µM 6b and 6d showed high inhibitory activity similarly to 1 µM all-trans retinoic acid (ATRA), indicating that the 2-thioxo group and length of two alkyl substituents at the 4-position are strongly related to activity.

Identification of mutations involved in the requirement of potassium for growth of typical Melissococcus plutonius strains.

Melissococcus plutonius is a fastidious honeybee pathogen, and the addition of KH(2)PO(4) to culture medium is required for its growth. Using genome sequences and a newly developed vector, we showed that mutations in genes encoding Na(+)/H(+) antiporter and cation-transporting ATPase are involved in the potassium requirement for growth.

Complete genome sequence of Melissococcus plutonius DAT561, a strain that shows an unusual growth profile and is representative of an endemic cluster in Japan.

We report the complete genome sequence of Melissococcus plutonius DAT561, which is a causative agent of European foulbrood. M. plutonius DAT561 is a representative of nonfastidious strains isolated in Japan. The addition of potassium phosphate was not required for normal growth, unlike for typical M. plutonius strain/isolates.

Complete genome sequence of Melissococcus plutonius ATCC 35311.

We report the first completely annotated genome sequence of Melissococcus plutonius ATCC 35311. M. plutonius is a one-genus, one-species bacterium and the etiological agent of European foulbrood of the honeybee. The genome sequence will provide new insights into the molecular mechanisms underlying its pathogenicity.

Skin Necrosis Due to the Extravasation of Irritant Anticancer Agents.

A 70-year-old man with malignant lymphoma was subjected to a fourth course of chemotherapy using gemcitabine and cisplatin. During the intravenous infusion of anticancer agents, pain and redness was observed at the site of insertion. The patient was subsequently treated with the strongest topical steroids and topical cooling agents. However, 2 weeks later, the affected area turned yellow, and the histopathological findings revealed skin necrosis of the entire dermis layer. It took two and a half months to cure the lesion. Close attention should be paid to the development of skin necrosis even when irritant anticancer agents such as gemcitabine and cisplatin are administered.

Rosehip inhibits xanthine oxidase activity and reduces serum urate levels in a mouse model of hyperuricemia.

Rosehip, the fruit of Rosa canina L., has traditionally been used to treat urate metabolism disorders; however, its effects on such disorders have not been characterized in detail. Therefore, the present study investigated the effects of hot water, ethanol and ethyl acetate extracts of rosehip on xanthine oxidase (XO) activity in vitro. In addition, the serum urate lowering effects of the rosehip hot water extract in a mouse model of hyperuricemia (male ddY mice, which were intraperitoneally injected with potassium oxonate) were investigated. Furthermore, the influence of rosehip hot water extract on CYP3A4 activity, which is the most important drug-metabolizing enzyme from a herb-drug interaction perspective, was investigated. Rosehip extracts of hot water, ethanol and ethyl acetate inhibited XO activity [half maximal inhibitory concentration (IC50) values: 259.6±50.6, 242.5±46.2 and 1,462.8±544.2 µg/ml, respectively]. Furthermore, the administration of 1X rosehip hot water extract significantly reduced the levels of serum urate at 8 h, which was similar when compared with the administration of 1 mg/kg allopurinol. Rosehip hot water extract only marginally affected CYP3A4 activity (IC50 value, >1 mg/ml). These findings indicate that rosehip hot water extract may present as a functional food for individuals with a high urate level, and as a therapeutic reagent for hyperuricemic patients.

Postnatal development of the mouse volatile papilla taste bud cells.

In the present study, we examined specific markers for taste bud cells in the mouse and the postnatal development of volatile papilla taste bud cells in ddY mice. We examined the immunoreactivity of 4 types of carbonic anhydrase isoenzymes, CA I, CA II, CA III and CA VI, as specific markers for taste bud cells, and K8.13 cytokeratin antibody as a specific marker for the lingual epithelial cells. Of the carbonic anhydrase isoenzymes, only CA III immunoreactivity was clearly detected in the spindle shaped gustatory cells. CA VI immunoreactivity was detectable in suspentacular cells. CA I and CA II antibodies did not recognize any taste bud cell specifically. K8.13 cytokeratin immunoreactivity was detected in the lingual epithelial cells, but not in taste bud cells. At 7 days after birth, the suckling phase, very small taste buds developed from the anaplastic gustatory cells. At 14 days after birth, the taste buds showed larger size than those at 7 days after birth. At 21 days birth, after the weaning phase, taste bud structure approximated the mature structure. These results demonstrate the specificity of anti-CA III and anti-CA VI for gustatory cells and suspentacular cells, respectively. These markers should be useful for an analysis of taste bud development in mice.

Typing of Melissococcus plutonius isolated from European and Japanese honeybees suggests spread of sequence types across borders and between different Apis species.

Melissococcus plutonius is an important pathogen of honeybee larvae and causes European foulbrood (EFB) not only in European honeybees (Apis mellifera) but also in other native honeybees. We recently confirmed the first EFB case in Japanese native honeybees (Apis cerana japonica) and isolated M. plutonius from this case. In this study, to obtain a better understanding of the ecology of M. plutonius and the epidemiology of EFB, we analyzed M. plutonius isolates that originated from European and Japanese honeybees in Japan using an existing multilocus sequence typing scheme. These analyzed Japanese isolates were resolved into six sequence types (STs), three of which were novel STs. Among these six STs, ST3 and ST12 were the two most common and found in isolates from both European and Japanese honeybees (or their environment). Moreover, these two STs were identified not only in Japan but also in other countries, suggesting the spread of some STs across borders and different honeybee species.

Development of duplex PCR assay for detection and differentiation of typical and atypical Melissococcus plutonius strains.

Melissococcus plutonius is the causative agent of an important honeybee disease, European foulbrood (EFB). In addition to M. plutonius strains with typical characteristics (typical M. plutonius), we recently reported the presence of atypical M. plutonius, which are phenotypically and genetically distinguished from typical M. plutonius. Because typical and atypical M. plutonius may have different pathogenic mechanisms, differentiation of these two types is very important for diagnosis and more effective control of EFB. In this study, therefore, a duplex PCR assay was developed to detect and differentiate typical and atypical M. plutonius rapidly and easily. On the basis of the results of comparative genomic analyses, we selected Na(+)/H(+) antiporter gene and Fur family transcriptional regulator gene as targets for detection of typical and atypical strains, respectively, by PCR. Under optimized conditions, the duplex PCR system using the designed primers successfully detected and differentiated all typical and atypical M. plutonius strain/isolates tested, while no product was generated from any other bacterial strains/isolates used in this study, including those isolated from healthy honeybee larval guts. Detection limits of the PCR were 50 copies of chromosome/reaction for both types, and it could detect typical and atypical M. plutonius directly from diseased honeybee larvae. Moreover, the duplex PCR diagnosed mixed infections with both M. plutonius types more precisely than standard culture methods. These results indicate that the duplex PCR assay developed in this study is extremely useful for precise diagnosis and epidemiological study of EFB.

Perineural xanthoma associated with type 2 diabetes mellitus and hyperlipidemia.

It is well known that patients with diabetes mellitus (DM) may demonstrate skin manifestations, or dermadromes, due to disease-related metabolic, vascular, neurologic, and/or immunologic disturbances; however, the pathogenesis of some of these manifestations remains unknown. Xanthomas often are associated with increased levels of serum cholesterol and/or triglycerides and therefore can present as a dermadrome in patients with a history of uncontrolled DM and hyperlipidemia. The presence of tender lesions in this patient population can indicate a diagnosis of perineural xanthoma. We report a case of perineural xanthoma arising in a patient with type 2 DM and hyperlipidemia.

IgA pemphigus with non-pustular erythematous lesions and IgA antibodies to desmocollins 1 and 2.

IgA pemphigus is a rare variant of pemphigus. IgA pemphigus is subdivided into intraepidermal neutrophilic IgA dermatosis-type (IEN-type), whose target antigen is still an enigma, and subcorneal pustular dermatosis-type, whose target antigen is desmocollin 1 (Dsc1). We report a 56-year-old Japanese male with IgA pemphigus showing atypical erythema. One month after erythema developed, the patient visited his private physician, and was tentatively diagnosed as having erythema multiforme. The patient had been intermittently treated with a low dose of oral prednisolone for a year without benefit before visiting our hospital. Clinical examination revealed irregularly-shaped and partially edematous erythema over the trunk and extremities without mucosal involvement. Neither bullae nor pustules were seen during the course. Direct immunofluorescence showed IgA deposition on cell surfaces of keratinocytes in the upper two thirds of the epidermis. Indirect immunofluorescence of monkey esophagus sections revealed IgA and IgG anti-cell surface antibodies. Our new enzyme-linked immunosorbent assays using eukaryotic recombinant proteins of human Dsc 1-3 detected IgA antibodies to Dsc1 and Dsc2. Although no apparent bullae were observed, the diagnosis of IgA pemphigus was made. Prednisolone 30 mg daily was required to control erythematous lesions. Although the pathomechanism for the unique skin lesion is unknown, the possibility that IgA pemphigus has a prodromal phase and that early administration of low dose prednisolone suppressed the development of pustules or bullae were considered.

Diversity of Melissococcus plutonius from honeybee larvae in Japan and experimental reproduction of European foulbrood with cultured atypical isolates.

European foulbrood (EFB) is an important infectious disease of honeybee larvae, but its pathogenic mechanisms are still poorly understood. The causative agent, Melissococcus plutonius, is a fastidious organism, and microaerophilic to anaerobic conditions and the addition of potassium phosphate to culture media are required for growth. Although M. plutonius is believed to be remarkably homologous, in addition to M. plutonius isolates with typical cultural characteristics, M. plutonius-like organisms, with characteristics seemingly different from those of typical M. plutonius, have often been isolated from diseased larvae with clinical signs of EFB in Japan. Cultural and biochemical characterization of 14 M. plutonius and 19 M. plutonius-like strain/isolates revealed that, unlike typical M. plutonius strain/isolates, M. plutonius-like isolates were not fastidious, and the addition of potassium phosphate was not required for normal growth. Moreover, only M. plutonius-like isolates, but not typical M. plutonius strain/isolates, grew anaerobically on sodium phosphate-supplemented medium and aerobically on some potassium salt-supplemented media, were positive for ß-glucosidase activity, hydrolyzed esculin, and produced acid from L-arabinose, D-cellobiose, and salicin. Despite the phenotypic differences, 16S rRNA gene sequence analysis and DNA-DNA hybridization demonstrated that M. plutonius-like organisms were taxonomically identical to M. plutonius. However, by pulsed-field gel electrophoresis analysis, these typical and atypical (M. plutonius-like) isolates were separately grouped into two genetically distinct clusters. Although M. plutonius is known to lose virulence quickly when cultured artificially, experimental infection of representative isolates showed that atypical M. plutonius maintained the ability to cause EFB in honeybee larvae even after cultured in vitro in laboratory media. Because the rapid decrease of virulence in cultured M. plutonius was a major impediment to elucidation of the pathogenesis of EFB, atypical M. plutonius discovered in this study will be a breakthrough in EFB research.

Isolated adrenocorticotropin deficiency associated with delirium and takotsubo cardiomyopathy.

We report a 65-year-old woman with isolated adrenocorticotropic hormone (ACTH) deficiency. The patient was transported to the emergency outpatient department by ambulance complaining of malaise and nausea. Because her laboratory data revealed hyponatremia, we performed endocrinological examinations and diagnosed isolated ACTH deficiency. After admission, she went into a delirious state and suffered from takotsubo cardiomyopathy due to adrenal insufficiency. Replacement therapy with hydrocortisone sufficiently improved her delirium and cardiomyopathy. We conclude that her unstable mental state and myocardial dysfunction were closely related to adrenal insufficiency and suggest that adrenal crisis may cause delirium and Takotsubo cardiomyopathy.