Six New Lanostane Triterpenoids from the Fruiting Body of Tyromyces sambuceus and Antiproliferative Activity.

During the search for secondary metabolites with antiproliferative activity, six new lanostane triterpenoids, tyrosamic acids A-F (1-6) together with ten known compounds (7-16), were isolated from the fruiting body of Tyromyces sambuceus. Their structures were elucidated using MS analyses, extensive 2D-heteronuclear NMR data interpretation and the structure of 3 was further confirmed by single-crystal X-ray data analyses. All lanostane triterpenoids (1-16) possesses a carboxy group at C-20 position and their strength of antiproliferative activity was affected by the presence or absence of a hydroxy group at C-15 position and at the side chain. Four of the compounds (1, 6, 10, 14) showed antiproliferative activities against human cancer cell lines with IC50 values of 16.8-48.3 µM (HL-60).

High cell density cultivation of recombinant Escherichia coli for hirudin variant 1 production.

A synthetic medium, TK-25, for high cell density cultivation (HCDC) of Escherichia coli K-12 was modified to support HCDC of strain JM109. By optimizing the culture conditions, the cell concentration of 65 g/l in 14 h was obtained in the optimized medium, namely TK-10, with glucose-fed batch cultivation. When these conditions were further applied for HCDC of E. coli JM109 harboring pUC-based recombinant plasmid which expresses a hirudin variant, HV-1-fused protein under the control of trp promoter, it grew to 24 g/l of dried cells expressed as an inclusion body as 15.9% of the total protein, corresponding to 1908 mg/l hirudin-fused protein.

Generation of muscle aquaporin 4 overexpressing transgenic mouse: its characterization at RNA and protein levels including freeze-fracture study.

We generated the muscle aquaporin 4 (AQP4) overexpressing transgenic mice in order to investigate the skeletal muscle pathology at RNA and protein levels. At RNA level, the AQP4 mRNA expression of soleus, EDL and cardiac muscles in Tg mice was statistically significantly higher than that in wild mice by the real-time reverse transcription polymerase chain reaction method. At protein level examinations, we used the immunoblot, immunohistochemistry and freeze-fracture electron microscopy. The immunoblot showed the single band of 31kDa with anti-AQP4 antibody in the extracts of soleus and EDL muscles of wild mice but not in extract of wild cardiac muscle; while the reaction band was noted in cardiac muscle of Tg mice and the reaction band was stronger in the extracts of soleus and EDL muscles of Tg mice. The immunohistochemistry showed that the expression of AQP4 at the myofiber surface of soleus and EDL muscles of Tg mice was more marked than that of wild mice and, interestingly, the AQP4 expression of these muscles of Tg mice appeared to be more remarkable in type 1 slow twitch myofibers as judged by the positive slow myosin immunostaining of adjacent serial sections. The immunofluorescence staining with anti-AQP4 antibody of cardiac muscles of wild mice revealed the scarcely immunopositive myofibers; whereas the immunostaining cardiac muscles of Tg mice contained the numerous AQP4 immunopositive myofibers. The freeze-fracture electron microscopy demonstrated that the orthogonal array densities in soleus and EDL muscle plasma membranes of Tg mice were significantly higher than those of wild mice and that the orthogonal array like particle density of cardiac muscle plasma membranes of Tg mice appeared to be more numerous than that of cardiac myofibers of wild mice. Finally the clinical phenotype of Tg mice appeared to be similar to that of wild mice. Further physiological examination with devices may suggest some about the physiological difference.

[Case of neuro-Behçet disease with homonymous quadrantanopsia due to an inflammatory lesion involving the lateral geniculate body].

We report a rare case of a 57-year-old woman of neuro-Behçet disease with homonymous quadrantanopsia due to an inflammatory lesion involving the lateral geniculate body. She had oral and genital ulcers since 1983, and uveitis since May 1985. She received diagnosis of incomplete Behçet disease and was prescribed cyclophosphamide since June 1985. After the treatment, she recovered completely from uveitis in July 1985. Painful subcutaneous nodules appeared in her right leg on June 21, 2004 and she had a high fever, headache and left visual disturbance on June 29, 2004. Therefore, she was admitted to our hospital on July 1, 2004. Physical and neurological examination showed erythema nodosum in the right lower extremity and left lower homonymous quadrantanopsia. Laboratory findings on admission showed leucocytosis, increased erythrocyte sedimentation rate and C-reactive protein, and positive HLA-B51. Cerebrospinal fluid analysis showed pleocytosis and a markedly high level of protein and interleukin-6. Brain magnetic resonance imaging (MRI) of T2-weighted images showed high intensity lesions in the circumference of the caudal thalamus, optic radiations, and right occipital cortex. T1-weighted images with gadolinium enhancement showed an enhanced lesion in the circumference of the right lateral geniculate body. From these results, she was diagnosed as having an acute relapsing phase of neuro-Behçet disease and she received steroid pulse therapy. Immediately after steroid pulse therapy, she received high-dose prednisolone which was gradually tapered. Brain MRI after treatment showed a high intensity lesion in the right lateral geniculate body. Homonymous quadrantanopsia remained nearly unchanged.

Aquaporin 1: examination of its expression and localization in normal human skeletal muscle tissue.

To examine aquaporin 1 (AQP1) expression in skeletal muscle tissue precisely, we performed reverse transcription-polymerase chain reaction (RT-PCR) at RNA level and immunoblot analysis, immunohistochemistry and immunoelectron microscopy at protein level. The RT-PCR study of total RNA from normal human skeletal muscle showed a strong single band of AQP1. At the protein level we used two commercially available antibodies, both of which recognize the cytoplasmic domain of the AQP1 molecule. One antibody gave positive results. Immunoblot of muscle extract showed a 30-kDa band protein, the molecular weight of which corresponded to that of AQP1. Immunohistochemically, AQP1 was immunostained at the myofiber surface both in type 1 and type 2 myofibers with almost the same intensity, and its staining pattern was rather diffuse and irregular compared with that of the anti-dystrophin antibody. The endomysial endothelial cells were also immunolabeled. Immunoelectron microscopy revealed that the immunogold particles indicating the presence of the AQP1 molecule were present along the inside surface of the muscle plasma membrane. However, another antibody showed negative results except for the endomysial endothelial cells which were positively stained. We drew the conclusion that AQP1 is expressed at the endomysial capillary endothelial cell and further AQP1 may be expressed at the human skeletal myofiber plasma membrane.

The utrophin promoter A drives high expression of the transgenic LacZ gene in liver, testis, colon, submandibular gland, and small intestine.

BACKGROUND: Duchenne muscular dystrophy (DMD) is caused by the absence of the muscle cytoskeletal protein dystrophin. Utrophin is an autosomal homologue of dystrophin, and overexpression of the protein is expected to compensate for the defect of dystrophin. The utrophin gene has two promoters, A and B, and promoter A of the utrophin gene is a possible target of pharmacological interventions for DMD because A-utrophin is up-regulated in dystrophin-deficient mdx skeletal and cardiac muscles. To investigate the utrophin promoter A activity in vivo, we generated nuclear localization signal-tagged LacZ transgenic mice, where the LacZ gene was driven by the 5-kb flanking region of the A-utrophin gene. METHODS: Four transgenic lines were established by mating four independent founders with C57BL/6J mice. The levels of mRNA for beta-galactosidase in several tissues were examined by RT-PCR. Cryosections from several tissues were stained with hematoxylin and eosin (H&E) and with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). RESULTS: The 5-kb upstream region of the A-utrophin gene showed high transcriptional activity in liver, testis, colon, submandibular gland, and small intestine, consistent with the endogenous expression of utrophin protein. Surprisingly, the levels of both beta-gal protein and mRNA for the transgene in cardiac and skeletal muscles were extremely low, even in nuclei near the neuromuscular junctions. These results indicate that the regulation of the utrophin gene in striated muscle is different from that in non-muscle tissues. CONCLUSIONS: Our results clearly showed that the utrophin A promoter is not sufficient to drive expression in muscle, but other regulatory elements are required.