Yutaka Tomoda

Identification of human placental leucine aminopeptidase as oxytocinase

Human placental leucine aminopeptidase (P-LAP) was purified from retroplacental serum for the first time by serial chromatography on columns of Matrex Blue A, DEAE-Sepharose CL-6B, phenyl-Sepharose 4B, chelating-Sepharose, and Sepharose CL-6B. The purified P-LAP was apparently homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the apparent molecular weight (Mr) was estimated to be 210,000. By comparing P-LAP activity with cystine aminopeptidase activity, we concluded that both activities were shared by the same molecule. We also examined the hydrolytic activity of P-LAP using naturally occurring peptide hormones and found that the enzyme hydrolyzed oxytocin, vasopressin, and angiotensin III. These results suggest that P-LAP shows oxytocinase activity and plays an important role in the regulation of the plasma level of these hormones during pregnancy.

Squamous cell carcinoma arising from mature cystic teratoma of the ovary: A clinicopathologic analysis

Objective There have been few studies concerning the clinical pathology of squamous cell carcinoma arising from ovarian mature cystic teratoma. Thus, the objective of this study is to determine clinicopathologic factors affecting survival in this rare tumor. Methods From September 1979 to June 1996, 37 patients with squamous cell carcinoma arising from ovarian mature cystic teratoma were treated by the Tokai Ovarian Tumor Study Group. A restrospective clinicopathologic and survival analysis of these patients was performed. The mode of infiltration was classified into expansive, moderately diffused, and diffused patterns. Results Although the 5-year survival rate was 94.7% and 80.0% for stage I and II patients, respectively, 12 and 13 patients with stage III died within 20 months (P = .0001). A significant difference was also observed between the survival of the groups with and without residual tumor at surgery (P = .0001). Pathologic features, grade, mode of infiltration, and vascular involvement were significant factors by univariate analysis. Multivariate analysis showed significant differences in survival related to grade (P = .0154) and mode of infiltration (P = .0053). The preoperative squamous cell carcinoma antigen level was significantly higher in the patients with squamous cell carcinoma arising from mature custic teratoma than in patients with mature cystic teratoma (P < .0001). Conclusion This study suggests that pathologic factors, grade, and mode of infiltration can provide valuable information for predicting the survival of patients with squamous cell carcinoma arising from mature cystic teratoma. In addition, squamous cell carcinoma antigen may be a useful marker to detect this disease preoperatively.

Phase II Study of Irinotecan and Cisplatin as First-Line Chemotherapy in Advanced or Recurrent Cervical Cancer

Irinotecan (CPT-11) and cisplatin are singly active against cervical cancer. We evaluated the efficacy and toxicity of CPT-11 plus cisplatin as first-line chemotherapy in patients with advanced or recurrent cervical cancer. Twenty-nine chemotherapy-naive patients with advanced or recurrent cervical cancer were treated with CPT-11 (60 mg/m2) on days 1, 8, and 15 by intravenous infusion over 90 min, followed by cisplatin (60 mg/m2 i.v.) on day 1 over 90 min. The patients’ median age was 57 years (range 35–75). Nineteen patients (66%) had advanced primary disease. Six patients with recurrent disease (21%) had been treated with prior radiotherapy. The remaining 4 patients (14%) had residual or recurrent disease after radical surgery. The histologic diagnoses were squamous cell carcinoma in 25 patients (87%), adenocarcinoma in 3, and adenosquamous cell carcinoma in 1. All eligible patients were included in the toxicity and response analysis based on the intent to treat. Two patients (7%) achieved a complete response and 15 (52%) a partial response (overall response rate: 59%, 95% confidence interval; 41–74%). Stable disease was recorded in 6 patients (21%) and progressive disease in 3 patients (10%). In 3 patients, image-guided evaluation of response was judged to be unfeasible at the time of independent extramural review (10%). The median time to response was 32 days (range 16–62 days). The median survival was 27.7+ months (range, 6.4–52.8+ months). Two dose-limiting side effects were observed: grade 3 (28%) or 4 (45%) neutropenia and grade 3 (7%) or 4 (7%) diarrhea. Other severe toxicities included anemia (45%), thrombocytopenia (3%), nausea/vomiting (31%), and alopecia (7%). The combination of CPT-11 with cisplatin is an active regimen for treatment of advanced or recurrent cervical cancer albeit with a significant degree of myelosuppression.

Effects of 17β-estradiol and progesterone on growth-factor-induced proliferation and migration in human female aortic smooth muscle cells in vitro

Objective: The significantly low prevalence of atherosclerotic cardiovascular disease in premenopausal women has been noted, and postmenopausal hormone replacement therapy (HRT) is associated with protection from this disease. The aim of this study was to investigate the effects of estrogen and progestin on growth-factor-induced vascular smooth muscle cell (VSMC) proliferation and migration in vitro. Methods: Two cell lines of human female aortic smooth muscle cells (AOSMCs) were used for the study. DNA synthesis was evaluated by [3H]thymidine incorporation into cells. Migration assay was performed using modified Boyden chambers. Results: The presence of estrogen receptors was determined by Western and Northern blot analyses. [3H]Thymidine incorporation into AOSMCs was induced by 10 ng/ml EGF alone, the combination of 10 ng/ml EGF with 2 ng/ml b-FGF-induced and 1 nM PDGF-BB alone. Migration of AOSMCs was induced by PDGF-BB. Since 17β-estradiol (E2, 10−9 M~ 10−6 M) inhibited the [3H]thymidine incorporation into AOSMCs stimulated by above mitogens and the 1 nM PDGF-BB-induced AOSMC migration in a dose-dependent manner, estrone (E1), estriol (E3) and progesterone (P) had no significant effects. The combination of P (10−9 M ~ 10−6 M) did not show any effect on these inhibitory effects of 10−7 M E2. Preincubation of AOSMCs with the anti-estrogenic agent, tamoxifen (10−6 M), significantly antagonized these inhibitory effects of 10−7 M E2. Conclusions: These findings suggest that the inhibitory effect of E2 on VSMC proliferation and migration might be one of the factors involved in the decreased incidence of atherosclerotic cardiovascular disease in premenopausal women and postmenopausal HRT, and P might not affect these estrogenic responses.

Surgical indications for resection in pulmonary metastasis of choriocarcinoma

One hundred and twenty‐two patients with choriocarcinoma were treated from 1965–1977. Pulmonary metastasis was noted in 82 of the 122 patients. In 21 cases, open thoracotomy and lobectomy were performed in conjunction with chemotherapy. Fifteen patients achieved complete remission and six patients died. It became evident that the factors with the greatest effect on the outcome of the surgical treatment are the preoperative hCG values and the extent of pulmonary metastasis. The following are the authors established criteria for the operative intervention of pulmonary metastasis of choriocarcinoma (modifying Thomfords principles): (1) The patient must be a good risk for surgical intervention. (2) That the primary malignancy is controlled, (the uterus has already been resected, or no angiographical evidence of tumor in pelvic cavity). (3) There is no evidence of metastatic disease elsewhere in the body. (4) Roentgenologic evidence of pulmonary metastasis is limited to one lung. (5) The urinary hCG value is below 1000 miU/ml.

Menotropin stimulation after prolonged gonadotropin releasing hormone agonist pretreatment for in vitro fertilization in patients with endometriosis

Two protocols were scheduled for in vitro fertilization and embryo transfer (IVF-ET) in patients with various stages of endometriosis who were resistant to conventional therapies. In the ultralong protocol (21 patients), gonadotropin releasing hormone agonist (Gn-RHa) was administered for at least 60 days prior to ovarian stimulation along with menotropin until human chorionic gonadotropin was injected. In the long protocol (11 patients), Gn-RHa was started at the midluteal phase and exogenous gonadotropin was commenced between the third and the seventh day of the menstrual cycle after pituitary suppression. The estradiol response and the number of retrieved oocytes, fertilized oocytes, cleaved oocytes, and transferred embryos were similar in both groups but the clinical pregnancy rate per transfer was superior in the ultralong protocol (67 vs 27%). The miscarriage rate was 14% (2/14) in the ultralong protocol. Prolonged Gn-RHa suppression of ovarian function before superovulation may overcome some causes of infertility in patients with endometriosis.

Increased mitochondrial damage by lipid peroxidation in trophoblast cells of preeclamptic placentas

Lipid peroxides and their related free radicals have been implicated in the pathogenesis of placental dysfunction in preeclampsia. Recent studies suggest that the placenta is a source of the increased lipid peroxides in the maternal circulation of women with preeclampsia. We examined intracellular localization of 4‐hydroxy‐2‐nonenal (HNE: a major aldehydic product of lipid peroxidation)‐modified proteins in human placentas by immunohistochemistry, and immunoblotting. The trophoblast layer of the chorionic villi showed intense immunoreactivity for HNE‐modified proteins in 4 of 12 preeclamptic placentas, whereas no staining was observed in 12 normal placentas. Immunoblotting revealed that three immunoreactive proteins with apparent molecular mass of 110 kDa, 75 kDa, and 70 kDa were localized in the mitochondrial fraction. The present results indicate that the damage to mitochondrial proteins by lipid peroxidation byproducts and subsequent dysfunction of trophoblasts contribute to the pathophysiology of preeclampsia.

Effects of placental proteases on maternal and fetal blood pressure in normal pregnancy and preeclampsia

Although many protease exist in human placenta, their physiologic roles are still unknown. Our study showed that placenta proteases metabolize vasoactive peptides possibly derived from the fetus. Because vasopressin and angiotensin are known to play an important role in normal and aberrant (preeclampsia) fetal-placental circulation, the clearance of these peptides in the placenta is important in controlling fetal blood pressure. Vasopressin and angiotensin act as a fetal-placental vasoconstrictor; therefore, placental proteases in human placenta are likely to work as a clearance factor for these peptides. Although human and animal pregnancy is normally associated with a refractory response to the pressor effect of exogenously infused angiotensin II, patients with preeclampsia, as well as nonpregnant women, are sensitive to the pressor effect of angiotensin II. Our study suggested that the decreased pressor responsiveness to angiotensin II in pregnancy is caused by increased inactivation of angiotensin II by angiotensinase in pregnant serum and the placenta. Although vasopressinase and angiotensinase activities increase with advancing gestation in normal pregnant sera, the activities of both enzymes in severe preeclampsia sera were clearly lower than those in normal pregnancy. Therefore, it is reasonable to speculate that the increased sensitivity to angiotensin II of preeclampsia is attributable to the decreased degradation of angiotensin II by placental angiotensinase. The negative correlations between the systolic to diastolic ratio obtained from pulsed Doppler measurement techniques and the activities of both enzymes in preeclampsia sera suggested that the systolic to diastolic ration, which reflected constriction of placental vessels, is influenced by the concentration of vasoactive peptides in the fetal-placental circulation due to changes in the activities of placental proteases. Placental proteases play important roles in controlling fetal and maternal blood pressure through regulation of the concentration of vasoactive peptides in the interface (placenta) between fetus and mother.

Prognostic factors in yolk sac tumors of the ovary. A clinicopathologic analysis of 29 cases

Twenty‐nine ovarian cancer patients with yolk sac tumors and germ cell tumors with yolk sac tissue as a component of their disease (16 endodermal sinus tumor, 11 mixed germ cell tumors, one embryonal carcinoma, and one polyembryoma) were treated with cytoreductive surgery and combination chemotherapy. Prognostic factors were investigated in this group. Patients with Stage I disease had a more favorable prognosis (P < 0.003) than those with Stages II and IV disease. The difference in prognosis was significant in cases where residual tumor was absent (P < 0.003) and in cases where ascites was either absent or less than 100 ml in volume (P < 0.05). Endodermal sinus tumor with either an intestinal (P < 0.05) or microcystic pattern (P < 0.01) was more common in survivors than in those who died. The age, preoperative serum alpha‐fetoprotein level, maximum tumor size, and tumor weight had no significant correlation with prognosis. In advanced cases, chemotherapy regimens including cisplatin gave better results than those containing vincristine, dactinomycin, and cyclophosphamide (P < 0.05). The optimal treatment of yolk sac tumors or tumors with yolk sac tissue as a component of the ovary is discussed in light of these results.

Expression of midkine and pleiotropin in ovarian tumors.

Objective To compare the expression of midkine and pleiotropin in malignant ovarian tumors with that in normal and benign ovarian tissue. Methods Total RNA was isolated from 23 samples of normal ovaries, 15 benign ovarian tumors, and 36 malignant ovarian tumors. Midkine and pleiotropin gene expression was examined by using Northern blot analysis. To confirm the localization of midkine expression, in situ hybridization and immunohistochemical analyses were performed. The truncated midkine messenger RNA was examined using polymerase chain reaction with complementary DNA synthesized from total RNA with reverse transcriptase. Results Expression of midkine gene was observed in 19 of 23 normal ovary samples and in 51 of 53 ovarian tumors (13 of 15 benign, both of the two borderline tumors, and all 36 malignant tumors). Pleiotropin gene was expressed in six normal ovaries and in 24 tumors (nine benign, two borderline, and 13 malignant tumors). The expression of midkine in germ cell tumors was significantly lower than in epithelial tumors, whereas expression in malignant epithelial tumors was significantly higher than in benign ones. In germ cell tumors, two samples with differentiated neural tissues showed high levels of pleiotropin gene expression. In situ hybridization and immunohistochemical analysis showed strong expression of midkine in cancer cells. The truncated midkine messenger RNA was not found in any of the normal, benign, or malignant tissues examined. Conclusion These results suggest an association between midkine and carcinogenesis. Expression of pleiotropin is more restricted, and high levels of its expression may be correlated with neural differentiation.