Cell-Free Synthesis of Human Endothelin Receptors and Its Application to Ribosome Display.

Engineering G-protein-coupled receptors (GPCRs) for improved stability or altered function is of great interest, as GPCRs consist of the largest protein family, are involved in many important signaling pathways, and thus, are one of the major drug targets. Here, we report the development of a high-throughput screening method for GPCRs using a reconstituted in vitro transcription-translation (IVTT) system. Human endothelin receptor type-B (ETBR), a class A GPCR that binds endothelin-1 (ET-1), a 21-residue peptide hormone, was synthesized in the presence of nanodisc (ND) composed of a phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG). The ET-1 binding of ETBR was significantly reduced or was undetectable when other phospholipids were used for ND preparation. However, when functional ETBR purified from Sf9 cells was reconstituted into NDs, ET-1 binding was observed with two different phospholipids tested, including POPG. These results suggest that POPG likely supports the folding of ETBR into its functional form in the IVTT system. Using the same conditions as ETBR, whose three-dimensional structure has been solved, human endothelin receptor type-A (ETAR), whose three-dimensional structure remains unsolved, was also synthesized in its functional form. By adding POPG-ND to the IVTT system, both ETAR and ETBR were successfully subjected to ribosome display, a method of in vitro directed evolution that facilitates the screening of up to 1012 mutants. Finally, using a mock library, we showed that ribosome display can be applied for gene screening of ETBR, suggesting that high-throughput screening and directed evolution of GPCRs is possible in vitro.

Umbilical cord presentation after use of a trans-cervical balloon catheter.

AIM: To examine the incidence of umbilical cord presentation, including cord prolapse (UCP) and cord descent (UCD), after the use of a trans-cervical balloon catheter (TCBC), such as a Foley catheter and a metreurynter, for the induction of labor (IOL). METHODS: A retrospective medical chart review was conducted, focusing on the occurrence of UCP and UCD in 800 women who underwent IOL with a TCBC at five hospitals during the study period (2008-2009 for two hospitals and 2006-2009 for three hospitals). The five hospitals had a total of 8245 deliveries during the study period. UCP and UCD were defined as the descent of the umbilical cord in advance of the presenting fetal part in the presence and absence of rupture of fetal membranes, respectively. RESULTS: The frequency of IOL using a TCBC with 70-250 mL of saline varied among the five hospitals from 4.9% to 18.8% (mean ± SD, 10.7 ± 5.0%). UCP and UCD occurred in two and four women, respectively, with the frequency of cord presentation varying among the hospitals from 0.0% to 1.8% (mean ± SD, 0.9 ± 0.9%); the cord presentation was significantly more likely to occur when 180-250 mL of saline was used, compared with when 70-150 mL of saline was used (8.2% [5/61] vs 0.15% [1/662], P < 0.0001). A change in the presenting fetal body part also occurred in 0.5% (4/800) of the women. CONCLUSION: The use of a TCBC with 180-250 mL of saline increases the risk of cord presentation.

A case of primary serous papillary carcinoma with unusual clinical presentation: distant lympho nodes metastasis without peritoneal dissemination.

CASE REPORT: A 50-year-old woman with primary serous papillary carcinoma (PSPC) of peritoneal origin located in the posterior uterine serosa and cul-de-sac without peritoneal dissemination. No peritoneal dissemination was detected but the tumor metastasized to para-aortic and supraclavicular lymph nodes. After first chemotherapy course, pericardial effusion occurred. A pericardiectomy was performed to prevent cardiac failure. Subsequent chemotherapy with paclitaxel and carboplatin was effective against this tumor CONCLUSION: In general, a typical type of PSPC usually develops distant metastasis with diffuse peritoneal dissemination; the present case shows unusual clinical behavior.

[Remarkably reduced tumor marker SCC levels by combined chemotherapy of paclitaxel and S-1 in two cases of advanced cervical cancer].

Case 1: A-35-year-old woman was diagnosed as cervical cancer Stage IIIb. When admitted to the hospital, her tumor marker SCC level was 50 ng/mL. Concurrent chemoradiation therapy was started on November, 2005. The SCC level was reduced by 0.9 ng/mL in February, 2006. In April, tumor recurrence was found by PET, and chemotherapy was restarted, but the SCC level was increased. In September, paclitaxel/S-1 therapy was performed, and the tumor markers were again reduced remarkably (SCC 9.8--> 1.3 ng/mL). Case 2: A-78-year-old woman was diagnosed as cervical cancer Stage IIIb. In August, 2004, concurrent chemoradiation therapy was started, and tumor markers were reduced (SCC 25.4--> 1.8 ng/mL). However, tumor markers were increased soon after the therapy. Chemotherapy was started, but it could not be maintained because of the side effects. In April, 2006, paclitaxel/S-1 therapy was performed, and the tumor markers were reduced remarkably (SCC 120--> 10 ng/mL). However, that therapy could also not be maintained because of the side effect. In July, she died of the cancer.

A phase I/II study of carboplatin and paclitaxel in patients with epithelial ovarian cancer.

BACKGROUND: This study was conducted to investigate the recommended dose of paclitaxel for use in combination with a fixed dose of carboplatin and to evaluate the toxicity and efficacy of carboplatin-paclitaxel combination chemotherapy in patients with epithelial ovarian cancer. METHODS: One hundred and ten patients were enrolled in the Phase I/II study and 97 patients were evaluated for further analysis, excluding 13 ineligible patients or patients with infringement of protocol: 15 patients for the Phase I and 82 for the Phase II study. In the Phase I trial, we studied dose escalation using a carboplatin dose of AUC 5 and paclitaxel levels of 150, 175 and 200 mg/m(2). The grades of toxicity of the regimen of all patients enrolled in the Phase II study (n = 82), the progression-free survival time (PFS) of optimal-debulked patients and complete responders (n = 62) and the response rate of suboptimal-debulked patients (n = 39) were investigated. RESULTS: After observing grade 4 neutropenia in four of six patients in the paclitaxel 200 mg/m(2) administration group, we chose 175 mg/m(2) as the recommended dose of paclitaxel in this regimen. At this dose, the median of PFS and response rate were 432 days (range, 19-907 days) and 66.7%, respectively. CONCLUSION: Combination chemotherapy using paclitaxel 175 mg/m(2) and carboplatin AUC 5 is very well tolerated and highly effective for the treatment of ovarian cancer.