Cell cycle control and CDC28/Cdc2 homologue and related gene cloning of Cryptococcus neoformans]

In Cryptococcus neoformans the DNA content of cells having tiny buds varied rather widely, depending on growth phases and strains used. Typically, buds of C. neoformans emerged soon after initiation of DNA synthesis in the early exponential phase. However, bud emergence was delayed to G2 during transition to the stationary phase, and in the early stationary phase budding scarcely occurred, although roughly half of the cells completed DNA synthesis. The timing of budding in C. neoformans was shifted to later cell cycle points with progression of the growth phase of the culture. Similarly, a deficit in oxygen was demonstrated to delay the timing of budding, prolong the G2 phase and cause accumulation of cells after DNA synthesis, but before commitment to budding. The C. neoformans homologue of the main cell cycle control gene CDC28/Cdc2 was isolated using degenerate RT-PCR. The full-length coding region was then amplified using primers to target the regions around the start and stop codons. The gene was called CnCdk1 and was found to have high homologies to S. cerevisiae CDC28 and S. pombe cdc2. To determine its function, its ability to rescue S. cerevisiae cdc28-temperature sensitive mutants was tested. S. cerevisiae cdc28-4 and cdc28-1N strains transformed with the pYES2-CnCdk1 construct exhibited growth at the restrictive temperature. Results of the sequence analysis and the ability of CnCdk1 to complement the S. cerevisiae cdc28-ts mutations support its assumed role as the CDC28/cdc2 homologue in C. neoformans.     

Drosera rotundifolia and Drosera tokaiensis suppress the activation of HMC-1 human mast cells

Ethnopharmacological relevance

Several Northern Hemisphere Drosera species have been used in the therapy of respiratory tract infections as the traditional medicine Droserae Herba.

Aim of the study

To determine the anti-inflammatory effects of Drosera species and to investigate a substitute material for Droserae Herba, we examined the effect of extracts of Drosera rotundifolia, Drosera tokaiensis and Drosera spatulata on activated T cell membrane (aTc-m)-induced inflammatory gene expression in HMC-1 human mast cells.

Materials and methods

Drosera rotundifolia, Drosera spatulata and Drosera tokaiensis were collected in Japan. Herbs were extracted with 80% EtOH, and subsequently applied to OASIS HLB column. HMC-1 cells were treated with each Drosera column-adsorbed fraction for 15 min, and subsequently added to aTc-m and incubated for 16 h. Inflammatory gene and protein expressions were determined by DNA microarray, RT-PCR and Western blotting.

Results

Drosera rotundifolia and Drosera tokaiensis fractions, but not the Drosera spatulata fraction, suppressed inflammatory gene expression induced by aTc-m in HMC-1 cells.

Conclusions

Drosera rotundifolia and Drosera tokaiensis suppressed activation of HMC-1 cells induced by aTc-m. Since the Drosera tokaiensis fraction was more effective than the traditionally used Drosera rotundifolia, Drosera tokaiensis is a likely substitute as a source of Droserae Herba.     

Rare associations of dens invaginatus and mesiodens.

Dens invaginatus is a developmental variation resulting from an alteration in the normal growth pattern of the dental papilla. Synonyms of this disturbance include dens in dente, invaginated odontome, tooth inclusion, and dentoid in dente. Radiographically, it is observed as infolding of a radiopaque ribbon-like structure, with equal density as enamel, extending from the cingulum into the root canal and sometimes reaching the root apex, assigning the appearance of a small tooth within the coronal pulp cavity. This article presents 2 case reports. The first describes an 8-year-old girl with dens invaginatus in a mesiodens; the second report describes a 16-year-old boy presenting with 2 mesiodens, both associated with dens invaginatus.     

Effect of gravitation for detection of bronchioloalveolar carcinoma on computed tomography

A 64-year-old female was found to have localized ground-glass opacity (GGO) in the middle lobe on a chest computed tomography (CT) for screening. Middle lobectomy with video-assisted thoracoscopic surgery (VATS) was undertaken, and pathological diagnosis was a bronchioloalveolar carcinoma (BAC) in stage IA. A follow-up CT a year following the surgery revealed localized GGO in area S⁶ of the left lung. However, it disappeared during the gravitation-dependent gradient in the observation period. The patient was scanned again under prone position to exclude the gravitational effect, resulting in definite detection of the GGO. Left extended S⁶ segmentectomy with VATS was performed, and pathological diagnosis was a BAC in stage IA. As GGO existing in a gravitation-dependent area may be masked by the gravitation-dependent density, a change of the scanning position may lead to a proper detection of the tumor for the diagnosis of BAC.     

4-Methylumbelliferone inhibits hyaluronate synthesis in human uterine cervical fibroblasts

AIM: Hyaluronate plays an important role in the regulation of cervical function during parturition. In our previous study we showed that 4-methylumbelliferone (MU) suppresses hyaluronate synthesis by cultured human skin fibroblasts. The present study investigated the effects of MU on fibroblasts obtained from the human uterine cervix and assessed the possibility of controlling cervical ripening with MU. METHODS: Human uterine cervical fibroblasts were collected from uterine cervices obtained from the uteri of three patients who had a total hysterectomy for uterine myoma at Hirosaki University Hospital. The fibroblasts were cultured in Dulbecco's modified Eagle's medium until confluence. They were then cultured in medium containing [3H]glucosamine (0.074 MBq/mL) with various MU doses. Hyaluronate synthesis was evaluated by assessing the incorporation of [3H]glucosamine into the soluble fraction of hyaluronate. Three independent studies were carried out on each specimen to clarify whether MU causes compositional changes or promotes hyaluronate degradation, whether the inhibitory effects of MU on hyaluronate synthesis are dose-dependent, and whether the effects of MU are reversible. RESULTS: MU added to the medium of the cultured cells reduced the synthesis of hyaluronate in a dose-dependent manner. After MU was removed from the medium, hyaluronate synthesis recommenced, and the amount of [3H]hyaluronate synthesized was similar to the control level. CONCLUSIONS: MU inhibits the synthesis of hyaluronate in human uterine cervical fibroblasts.     

Distribution characteristics of immunosuppressants FK506 and cyclosporin A in the blood compartment

Using two representative immunosuppressants, FK506 (FK) and cyclosporin A (CyA), of which the mechanism of pharmacological action is the same although there is a great difference in the pharmacological intensity, the distribution characteristics were studied in both in vivo and in vitro experiments using rat, dog, and human blood. Blood samples were fractionated by means of sedimentation in Ficoll-Paque®, and the drug contents in the diluted plasma fraction, erythrocyte fraction, and lymphocyte fraction were measured by an HPLC method. FK distributes to the lymphocyte fraction to a level about three times greater than that of CyA, while CyA distributes to the erythrocyte fraction to a level ten times that of FK. The distribution pattern of these fractions was independent of the drug concentration and species after correcting the drug concentration in each fraction with the blood drug concentration. The uptakes of FK and CyA in the isolated lymphocytes obtained from the rat spleen and human peripheral blood were also studied. The amount of FK taken up by the spleen lymphocytes is five times greater than that of CyA. In the case of the uptake study using human peripheral blood lymphocytes, the concentration of FK in the lymphocyte is 100-fold higher than that of CyA. This difference in the lymphocyte level between the two immunosuppressants is thought to be one of the reasons why FK is more potent than CyA, a difference of about 100-fold in the in vitro pharmacological study and about tenfold in the in vivo organ transplantation experiments.     

Development of new immunoradiometric assay for CA 125 antigen using two monoclonal antibodies produced by immunizing lung cancer cells

CA 125 is an antigen associated with non-mucinous epithelial ovarian cancer, which is defined by OC 125 antibody developed by immunizing ovarian cancer cells. We have produced two monoclonal antibodies, 130-22 and 145-9, by using the human lung adenocarcinoma cell line PC-9. Both 130-22 and 145-9 antibodies recognized CA 125 antigen. However, the binding sites seemed to be separate from those of OC 125. Testing by 9 immunoradiometric assays (IRMA), using different combinations of the 3 monoclonal antibodies 130-22, 145-9 and OC 125 demonstrated that the best standard curve for detecting CA 125 could be obtained by a "simultaneous sandwich" assay based on a mixture of 125I-labeled OC 125 and 130-22 or 145-9 coated beads. One-step IRMA, using 130-22 as a tracer and 145-9 as an immunoadsorbent, also showed good reproducibility and sensitivity for measuring CA 125. Antigens were detectable in the culture supernatants of PC-9 cells and 5 of 6 ovarian cancer and endometrial adenocarcinoma cells. These results indicate that one-step IRMA using 130-22 and 145-9 is useful for detecting CA 125 antigen.