Biochemical characterization of galloyl pedunculagin (ellagitannin) as a selective inhibitor of the beta-regulatory subunit of A-kinase in vitro.

The inhibitory effects of galloyl pedunculagin (GP) and eugeniin on the phosphorylation of histone H2B by cAMP-dependent protein kinase (A-kinase) and autophosphorylation of its beta-regulatory subunit (A-kinase beta) were examined in vitro. It was found that (i) GP (ID(50) = approx. 50 nM) effectively inhibits the activity of A-kinase (heterodimer), but high doses are required to inhibit the activities of the alpha-catalytic subunit (ID(50) = approx. 0.25 microM) and casein kinase II (CK-II, ID(50) = approx. 0.6 microM); (ii) GP inhibits the autophosphorylation of A-kinase beta in a dose-dependent manner with an ID(50) of approx. 6.6 nM, which is about 30-fold lower than that observed with CK-II beta; and (iii) GP reduces the suppressive effect of the beta-subunit on the activity of the alpha-subunit. In addition, purified bovine heart A-kinase precipitates when incubated with excess GP at pH 5.0. A similar precipitation of A-kinase was observed with eugeniin. These results show that the direct binding of GP to the beta-subunit prevents the physiological interaction between the beta- and alpha-subunits of A-kinase in vitro. This conclusion is presumably consistent with the binding affinity of proline-rich proteins with tannins, since A-kinase beta contains a proline-rich domain that interacts with GP or eugeniin. Therefore, GP will serve as a powerful inhibitor for in vitro and in vivo cellular studies of A-kinase beta-mediated signal transduction.

A retrospective survey of clinical, pathologic, and prognostic features of adnexal masses operated on during pregnancy.

OBJECTIVES: To evaluate retrospective data concerning patients with adnexal masses that were managed surgically during pregnancy and their effect on fetal outcome. METHODS: Data were reviewed concerning pregnant women who required surgery at our hospital between 1980 and 1997 for an adnexal mass. RESULTS: In the past 19 years at our hospital a total of 69 Japanese women aged 28.5 +/- 3.4 years (including 2 women with twin pregnancies) were diagnosed with adnexal masses that required surgery. The masses (10.2 +/- 4.5 cm in the largest diameter) were removed at 13.9 +/- 3.7 weeks of gestation. The pathologic features of the 69 lesions were as follows: 33 mature cystic teratomas, 13 functional cysts, 8 mucinous cystadenomas, 6 endometriotic cysts, 4 paraovarian cysts, 3 serous cystadenomas, and 2 malignant neoplasms. Of the 60 patients for whom the outcome of pregnancy was available, 7 (12%) gave birth before 37 weeks of gestation, while 2 (3.3%) experienced spontaneous abortions. There were 3 perinatal deaths among the 60 infants. Two of these 3 infants died due to major anomalies. CONCLUSIONS: Although larger studies are required for confirmation, our results suggest that an adnexal mass might be associated with an adverse fetal outcome. Surgical intervention at < 24 weeks of gestation per se might not have been related to the adverse outcomes. We emphasize that surgical intervention during pregnancy can be avoided in patients who have ultrasonographically pathognomonic features of benign cystic teratomas, which are the most common neoplasms operated on during pregnancy.

Risk of premature birth in multifetal pregnancy.

The risk of preterm delivery (< 37 weeks of gestation) is approximately nine times higher in women with multifetal pregnancies than in women with singleton pregnancies. However, it is possible that the risk will vary according to gestational week. To assess the risk of premature birth within 1 week by gestational age among multifetal pregnancies and compare the estimated risk with that of singleton pregnancies, we analyzed 6,036,475 infants born in singleton pregnancies and 90,887 infants born in multifetal pregnancies in Japan (> or =22 weeks) over the 5-year period 1989-1993. An estimate of the risk of birth within 1 week at gestational week n was obtained by dividing the number of infants delivered at gestational week n by the number of infants delivered at or beyond gestational week n. The risk at 22 weeks was 0.9 per 1000 fetuses for singleton pregnancies and 5.0 per 1000 for multifetal pregnancies. The risk remained relatively stable until 27 weeks of gestation, then sharply increased toward 36 weeks of gestation in both singleton and multifetal pregnancies. The odds ratio for birth within 1 week for fetuses of multifetal pregnancies compared with fetuses of singleton pregnancies was 5.9 (95% CI, 5.4-6.5) at 22 weeks of gestation, increasing gradually with increasing gestational age until 33 weeks of gestation (13.7; 95% CI, 13.1-14.2) but declining thereafter to 8.8 (95% CI, 8.6-8.9) at 36 weeks of gestation. Results of data analysis for each year of the 5-year period did not differ substantially.

Biochemical characterization of 60S acidic ribosomal P proteins from porcine liver and the inhibition of their immunocomplex formation with sera from systemic lupus erythematosus (SLE) patients by glycyrrhizin in vitro.

The three casein kinase II (CK-II) phosphate acceptors (p35, p17 and p15) in the Superdex CK-II fraction prepared from a 1.5 M NaCl extract of porcine liver were selectively purified by glycyrrhizin (GL)-affinity column chromatography (HPLC) as a heterocomplex associated with CK-II. Determination of the N-terminal amino acid sequences and immunological tests confirmed that these three CK-II phosphate acceptors belong to the family of 60S acidic ribosomal proteins (P0, P1 and P2). Three polyphenol-containing anti-oxidant compounds [catechin, epigallocatechin gallate (EGCG) and quercetin] inhibited CK-II activity (phosphorylation of these ribosomal P proteins) in a dose-dependent manner in vitro. Quercetin (ID50 = approx. 50 nM) was found to be an effective CK-II inhibitor. In contrast, CK-II activity was significantly stimulated by lower doses (0.3-3 microl) of GL, but was inhibited at high doses above 30 microM. As expected, GL at high doses above 200 microM inhibited the immunocomplex formation of 60S acidic ribosomal P proteins with their specific antibodies in the sera from patients with systemic lupus erythematosus (SLE). These results suggest that (i) a GL-affinity column is useful for effective purification of 60S acidic ribosomal P proteins from various mammalian cells as a heterocomplex associated with CK-II; and (ii) a relative high dose of GL may prevent the immunocomplex formation of 60S acidic ribosomal P proteins with their specific antibodies in the sera of SLE patients.

Severe hypoproteinemia in a fetus after pleuro-amniotic shunts with double-basket catheters for treatment of chylothorax.

The prognosis of a fetus with hydrothorax at mid-trimester is extremely poor. We encountered a fetus who developed bilateral chylothoraxes at 23 weeks of gestation. Bilateral pleuroamniotic shunts with double-basket catheters were successfully installed at 25 weeks of gestation. Hydrothorax did not recur in this fetus. After the shunting, however, polyhydroamnios, fetal hypoproteinemia, and placental edema developed, and the hydrops worsened. The drainage of the fetal pleural effusion into the amniotic cavity was believed to have contributed to these complications. The infant, born at 29 weeks of gestation, died of cardiac failure and pulmonary hypoplasia. Thus, the shunts did not ameliorate the adverse conditions in this patient.

Influence of chorioamnionitis on survival and morbidity in singletons live-born at < 32 weeks of gestation.

BACKGROUND: Chorioamnionitis (CAM) may accelerate lung maturation in fetuses. It is possible that CAM prevents infant death after live birth. METHODS: A retrospective study of live-born singletons at < 32 weeks of gestation between 1993 and 1997. Perinatal risk factors for adverse outcomes were analyzed using a logistic regression model, with special reference to the presence of histologically confirmed CAM. Adverse outcomes included infant death before 1 year of age, and survival with cerebral palsy and/or mental retardation. RESULTS: A total of 81 infants, weighing 1181 +/- 426 g, were born at 28.1 +/- 2.3 weeks of gestation. Of those, 15 (19%) died before 1 year of age, while 16 (20%) infants developed major handicaps by 1.5 years of age (six with cerebral palsy, eight with mental retardation, and two with both cerebral palsy and mental retardation). CAM, present in 44 women, was significantly associated with a reduced risk of death after live birth, with an odds ratio of 0.11 (p = 0.01). Only the presence of such intracranial lesions as periventricular leukomalacia and intraventricular hemorrhage were significantly associated with an increased risk of major handicaps (odds ratio of 11.0, p = 0.04). Adverse outcomes occurred in a similar proportion of infants in groups without CAM (14/37) and with CAM (17/44). However, among infants with adverse outcomes, the number of deaths was significantly higher in the group without CAM (10/14) vs. with CAM (5/12) (p < 0.05). CONCLUSIONS: The presence of CAM may somehow prevent infant death after live birth. Larger studies are required to confirm this phenomenon.

Uterine leiomyoma in pregnancy: its influence on obstetric performance.

OBJECTIVE: To assess the effects of uterine leiomyoma on obstetrical performance. METHODS: We reviewed the medical records of 102 women with singleton pregnancies who were found ultrasonographically to have uterine leiomyomas during the first half of their pregnancy and who gave birth at our hospital at > or = 22 weeks of gestation between January 1990 and December 1997. RESULTS: The 102 women gave birth to 101 healthy infants, weighing 2,974 +/- 579 g at 38.8 +/- 2.6 weeks of pregnancy. One woman experienced an unexplained antepartum fetal death at 24 weeks of gestation. Bleeding at the first trimester occurred in 16% of the women. Pain localized in the lower abdomen and requiring relief occurred in 28% of the women during the first or second trimester. Tocolytic treatment was required in 25% of the pregnancies, and preterm delivery occurred in 12% thereof. A cesarean section was performed in 39% of the pregnancies. Bleeding > or = 500 ml occurred at delivery in 48% of the cases. The largest fibroid, > 6 cm in diameter, which was seen in 51 women, was associated with higher frequencies of tocolytic treatment (41%), preterm delivery (24%), bleeding > or = 500 ml at delivery (59%), and cesarean delivery (51%). In 76 women (75%) who attempted vaginal delivery, the obstetrical outcome was comparable to that of 115 control women who were matched regarding age, parity, and gestational week. CONCLUSIONS: Although pain in the lower abdomen, the requirement of tocolytic treatment, preterm delivery, and cesarean delivery were common, the neonatal outcome was fairly good in women with uterine leiomyomas. The present data might be encouraging to pregnant women with uterine leiomyomas.

Biochemical characterization of 60S acidic ribosomal P proteins associated with CK-II from bamboo shoots and potent inhibitors of their phosphorylation in vitro.

Three effective phosphate acceptors (35, 15 and 13 kDa polypeptides) for casein kinase II (CK-II) in the Superdex CK-II fraction prepared from a 0.5 M NaCl extract of bamboo shoots were selectively purified by glycyrrhizin (GL)-affinity column chromatography (HPLC). These three proteins (p35, p15 and p13) were identified as 60S acidic ribosomal P proteins by determination of their partial N-terminal sequences. CK-II was associated with p35 since the GL-affinity fraction was coprecipitated with an anti-serum against Drosophila CK-IIbeta. Moreover, a derivative (oGA) of glycyrrhetinic acid (GA) and several polyphenol-containing anti-oxidative compounds [quercetin, epigallocatechin gallate (EGCG) and two isoflavones, i.e., 3',4',7-trihydroxyisoflavone (3',4',7-THI) and 8-chloro-3',4',5,7-tetrahydroxyisoflavone (8C-3',4',5,7-THI)] inhibited the CK-II-mediated phosphorylation of 60S acidic ribosomal P proteins in vitro. Quercetin was found to be the most effective compound on CK-II activity since its ID50 was approx. 50 nM. These results suggest that (i) GL-affinity column chromatography is useful for the selective purification of 60S acidic ribosomal P proteins as a heterocomplex associated with CK-II from various cell sources; (ii) natural anti-oxidative compounds with polyphenols, but not GL and GA, act as potent CK-II suppressors; and (iii) CK-II mediates the regulation of the physiological functions of 60S acidic ribosomal P proteins in growing plant cells.

Emergency cervical cerclage: relation between its success, preoperative serum level of C-reactive protein and WBC count, and degree of cervical dilatation.

OBJECTIVE: To assess the relation between the preoperative serum level of C-reactive protein (CRP) and the WBC count and the efficacy of emergency cervical cerclage. STUDY DESIGN: We retrospectively reviewed the medical records of 17 women (16 singleton pregnancies and 1 twin pregnancy) who underwent emergency cervical cerclage (McDonald technique) between 21 and 26 weeks of gestation. The uterine cervix was dilated >/=3.0 cm and the intact (not ruptured) fetal membranes were visible or protruded into the vagina in all patients. The serum level of CRP and the WBC count were determined preoperatively and postoperatively. Emergency cervical cerclage was considered successful if delivery occurred >/=14 days after the procedure. RESULTS: Emergency cervical cerclage was successful in 12 women, including the woman with a twin pregnancy, but failed in 5 women. The preoperative serum level of CRP and the WBC count were significantly lower and dilatation of the cervix was significantly less in the success group than in the failure group. Cerclage was successful in all eight patients with cervical dilatation 4.0 cm (p < 0. 05). Cerclage was successful in all 11 women with a preoperative serum level of CRP 4.0 mg/dl or a WBC count >14,000/ microl (p < 0.01). CONCLUSION: A preoperative CRP value

Evidence for two distinct cross-bridge populations in tetanized frog muscle fibers stretched with moderate velocities.

When tetanized frog skeletal muscle fibers are subjected to moderate-velocity stretches (< 1 L0/s), the tension developed above the level of isometric tension starts to decay after a sudden reduction of stretch velocity by more than 40-50%, though the fibers are still being stretched. We analysed the decay of tension response caused by the sudden reduction of stretch velocity, by applying three different types of stretch to a tetanized fiber, i.e., a 1.5% stretch with velocity V1 (stretch-1), a 1.5% stretch with velocity V2 < V1 (stretch-2), and a 3% stretch consisting of stretch-1 and stretch-2 applied in succession (stretch-3) and comparing the corresponding tension responses, TR-1, TR-2, and TR-3. It was found that TR-3 to stretch-3 resulted from algebraical summation of TR-1 to the preceding stretch-1 and TR-2 to the subsequent stretch-2. These results can be accounted for by assuming two distinct cross-bridge populations in stretched fibers.

Ovarian mixed germ cell tumor composed of dysgerminoma, endodermal sinus tumor, choriocarcinoma and mature teratoma in a 44-year-old woman: case report and literature review.

A case of ovarian mixed germ cell tumor in a 44-year-old woman was examined. The tumor was well circumscribed, measured 15 x 11 x 10 cm and appeared solid and partly cystic on the cut surface. Light microscopic examinations revealed that the tumor was composed of four different neoplastic germ cell elements, intermingled with each other. They are: (i) choriocarcinoma, immunohistochemically positive for human placental lactogen (hPL) and human chorionic gonadotropin (hCG); (ii) dysgerminoma, positive for placental alkaline phosphatase; (iii) endodermal sinus tumor positive for alpha-fetoprotein (AFP); and (iv) mature teratoma. Among these histological types, dysgerminoma occupied more than 50% of the neoplasm. The patient was diagnosed as a stage la ovarian mixed germ cell tumor and was subsequently treated with chemotherapy. A second-look laparotomy after completion of chemotherapy revealed no residual tumors in the abdomen and the patient is alive and well 15 months after operation. This is the fourth reported case of ovarian mixed germ cell tumor arising in patients over 40 years old.

Evidence for bidirectional functional communication between myosin subfragments 1 and 2 in skeletal muscle fibers.

It has been shown that, in glycerinated skeletal muscle fibers, polyclonal antibody directed against myosin subfragment 2 (S-2) eliminates Ca(2+)-activated isometric force development, while Mg-ATPase activity of the fibers remains unchanged. To further explore possible involvement of myosin S-2 in muscle contraction, we studied the effect of anti-S-2 antibody on rigor linkage formation in the fibers. It was found that the antibody inhibited development of rigor force in a dose- and time-dependent manner, without changing the relation between muscle fiber stiffness and force. If, however, the antibody was applied after development of rigor force, it had no effect on both the stiffness and force. These results strongly suggest bidirectional functional communication between myosin subfragments 1 and 2 in the fibers.

Force-velocity relation of sliding of skeletal muscle myosin, arranged on a paramyosin filament, on actin cables.

To investigate in vitro ATP-dependent sliding of regularly arranged myosin molecules on actin filaments, we prepared thick hybrid filaments in which myosin molecules isolated from rabbit skeletal muscle were arranged around the paramyosin core (length, 10-20 micron; diameter,

Evidence for two distinct cross-bridge populations in tetanized frog muscle fibers stretched with moderate velocities.

When a tetanized frog skeletal muscle fiber is stretched with moderate velocities (< 1 L0/S), the tension developed above the level of isometric tension starts to decay after a sudden reduction of stretch velocity by more than 40-50%, though the fiber is still being stretched. We analyzed the decay of tension responses caused by the sudden reduction of stretch velocity, by applying three different types of stretch, i.e. a 1.5% stretch with velocity V1 (stretch 1), a 1.5% stretch with velocity V2 < V1 (stretch 2), and a 3% stretch consisting of stretches 1 and 2 applied in succession (stretch 3) and comparing the corresponding tension responses, TR 1, TR 2 and TR 3. It was found that TR 3 to stretch 3 was equal to algebraical sum of TR 1 to the preceding stretch 1 and TR 2 to the subsequent stretch 2. In other words, TR 2 started on the falling tension baseline equal to the decay of TR 1 after completion of stretch 1, These results can be explained by assuming two distinct cross-bridge populations mechanically acting in parallel with each other.

Bidirectional functional communication between myosin subfragments 1 and 2 in skeletal muscle fibers.

It has been shown that polyclonal antibody directed against myosin subfragment 2 (S-2) eliminates Ca(2+)-activated isometric force development in glycerinated skeletal muscle fibers while Mg-ATPase activity of the fibers remains unchanged. To further explore possible involvement of myosin S-2 in muscle contraction, we studied the effect of anti-S-2 antibody on rigor linkage formation in the fibers, and found that the antibody inhibited development of rigor force in a dose- and time-dependent manner without changing the relation between muscle fiber stiffness and force. If, however, the antibody was applied after development of rigor force, it had no effect on both the stiffness and force. These results strongly suggest bidirectional functional communication between myosin subfragments 1 and 2 in the fibers.

Diagnostic use of cordocentesis in twin pregnancy.

We performed percutaneous umbilical blood sampling, under ultrasound guidance, in both twins of 11 twin pregnancies in whom twin-to-twin transfusion syndrome was suspected. Chorionicity and the presence of placental vascular anastomoses were assessed postnatally. The hemoglobin concentration, hematocrit and other biochemical variables in fetal blood were evaluated in both twins. The mean hemoglobin difference between the large and small twins was 4.8 g/dl (range 1.8-8.1 g/dl) in 5 monochorionic discordant twin pairs, and 1.2 g/dl in a monochorionic twin pair without discordancy. Hemoglobin values did not differ between dichorionic twins. The mean hematocrit difference between monochorionic discordant twins was 18.3%. In dichorionic discordant twins, the inter-twin hematocrit difference was very small. Total protein and albumin were normal in all twins.

[Hematological values in fetal blood by cordocentesis].

Reference ranges for four hematological parameters--hemoglobin, hematocrit, white blood cell and platelet count--in each gestation were established from values for fetal blood samples obtained by cordocentesis from 72 pregnancies at 16-39 weeks gestation. These 72 fetuses turned out as normal or single malformations which should not affect fetal hematological values. Significant correlations were observed between hemoglobin, hematocrit and platelet counts and the number of weeks of gestation. No correlation was found between the white blood cell count and gestational age. Fetal anemia (hematocrit was below the lower limit of the 95% confidence interval) was found in 18 (7%) of all the fetuses that underwent cordocentesis, including nonimmune hydrops fetalis (8 cases), Rh isoimmunization (4 cases), twin-to-twin transfusion syndrome (3 cases), trisomy 18 (2 cases) and autoimmune thrombocytopenia (1 case). Of 26 hydropic fetuses, 8 (31%) were anemic. The prognosis of those fetuses depended on either the gestational age or the severity of the fetal anemia. Our results are useful in diagnosing fetal hematological disorders and to make decisions for fetal therapy.

[Incidence of fetal chromosomal aberration in prenatal cytogenetic examination].

One thousand forty-seven fetal samples were obtained from women who received the prenatal cytogenetic examination for different reasons. In this study, the incidence of fetal chromosomal aberration related to each reason was analyzed. The incidence of de novo chromosomal aberration in the fetuses of the higher maternal age group was 1.5% (8/525: 4 cases of 21-trisomy, 2 cases of 18-trisomy and 2 cases of 47,XXY). The incidence in the group of women who had borne (a) chromosomally abnormal child(ren) was 0.5% (1/202). There was no repeat of 21-trisomy, 18-trisomy or 13-trisomy in this study. The incidence in the group of women who had ultrasonographic abnormalities was 16.9% (26/154). In this study, abnormal amniotic fluid volume, intrauterine growth retardation, malformation, hydrops fetalis, intestinal obstruction, omphalocele, single cord artery and cerebellar hypoplasia were related to chromosomal aberrations. The incidence in the group of women whose husband or herself was cytogenetically abnormal was 40.0% (26/65). This high value indicated that parental translocation and inversion are easily transmitted to their offspring.