Development of a time-resolved fluorometric detection system using diffusion-enhanced energy transfer

A novel detection system using both emission energy transfer and time-resolved fluorometry (TRF) was developed, with a europium chelate as the energy donor and a novel fluorophore SNR1, excitable with long-wavelength light corresponding to europium emission, as the energy acceptor. When the donor and acceptor molecules were mixed in solution, energy transfer was observed without direct attachment of the donor and the acceptor, via a diffusion-enhanced energy-transfer mechanism. Thus, the acceptor emission can be detected as a long-lifetime fluorescence in TRF. When the fluorescence properties of the acceptor molecule are changed by interaction with an enzyme or other bioactive molecule, the change can be detected as a long-lived sensitized emission. If we develop or select suitable acceptor molecules, this simple and convenient system should be applicable to a wide variety of bioactive molecules. Since it is based on TRF, it can be used for high-resolution assay.

Umbilical cord ulceration and intestinal atresia.

Umbilical ulceration is an extremely rare complication in the perinatal period. We encountered a case of intestinal atresia complicated by massive intrauterine hemorrhage due to the umbilical cord ulceration. This is the fifth reported case demonstrating an association between the umbilical cord ulceration and intestinal atresia.

A cloverleaf skull syndrome probably of Beare-Stevenson type associated with Chiari malformation.

A case of cloverleaf skull (CLS) syndrome with Chiari malformation was reported. The patient developed congenital hydrocephalus, upper airway obstruction and breath holding spells. Ventriculo-peritoneal shunt improved the hydrocephalus, but the patient progressively developed apneic episodes in spite of intubation. Brain magnetic resonance imaging (MRI) disclosed severe Chiari malformation. Laminectomy of the cervical vertebrae and craniectomy at the occipital bone were performed to relieve the brain stem compression. Postoperatively, the respiratory symptoms improved slightly, but 8 weeks later the patient suddenly died. Literature survey revealed that this is the second case report of CLS associated with Chiari malformation. It was assumed that the respiratory problems in our patient are ascribable to the Chiari malformation as well as the upper airway obstruction. The patient here reported seems to be a case of Beare-Stevenson cutis gyrata syndrome, in view of an association of multiple characteristic dysmorphic features, in addition to CLS.

Perinatal intracranial hemorrhage due to severe neonatal alloimmune thrombocytopenic purpura (NAITP) associated with anti-Yukb (HPA-4a) antibodies.

Neonatal alloimmune thrombocytopenic purpura (NAITP) is one of the causes of thrombocytopenia in the newborn period. The thrombocytopenia is caused by maternal transplacental antiplatelet alloantibodies. We report a case of NAITP in a newborn infant having subarachnoid hemorrhage. Examination of platelet antibodies revealed anti-Yukb, that is, human platelet antigen (HPA)-4a incompatibility. Cranial ultrasound and brain magnetic resonance imaging revealed subarachnoid hemorrhage in the temporal region inferior to the cephalohematoma. The lesion seemed to have been sustained during delivery. The patient was treated with high-dose gamma-globulin and several transfusions of random donor platelets and showed a good clinical course. This is the second reported case of NAITP associated with the Yuk antigen system having intracranial hemorrhage.

Unreliability of platelet glucose-6-phosphatase for the diagnosis of glycogen storage disease type Ia.

The diagnosis of glycogen storage disease type Ia currently uses enzyme analysis of liver tissue. This requires liver biopsy in the at-risk neonate or fetus. Conflicting reports have appeared in the literature on the use of peripheral platelet glucose-6-phosphatase activity for the diagnosis of this disorder. We have applied a sensitive radiometric assay system to the measurement of glucose-6-phosphatase activity in peripheral platelets. Two families with affected members were analysed, revealing no differences in glucose-6-phosphatase activity as compared with control values. Platelet measurement of glucose-6-phosphatase does not appear to be useful for the diagnosis of glycogen storage disease type Ia.

[Evaluation of the renal artery in the fetuses with growth retardation and oligohydramnios by two dimensional Doppler ultrasonography].

Renal blood flow dynamics were studied with color and pulsed Doppler ultrasound in 102 normal fetuses at 20 to 40 weeks of gestation, 11 growth retarded fetuses (group A), 15 fetuses with oligohydramnios (group B) and 10 growth retarded and oligohydramniotic fetuses (group C) at 32-40 weeks of gestation. The maximum systolic velocity (VMAX), early diastolic peak velocity (VEA), end diastolic velocity (VED) and resistance index (RIEA:VMAX-VEA/VMAX and RIED:VMAX-VED/VMAX) were measured in the renal artery. VMAX was also measured in the ascending aorta (AO) and pulmonary artery (PA). In the normal fetuses, blood flow velocity increased with gestational age, but VMAX and VEA remained constant after 28 weeks of gestation. The resistance index decreased with gestational age, but RIEA remained constant after 28 weeks of gestation. RIED in groups A and C increased significantly, and VMAX in groups B and C, and VED in group C decreased significantly compared with normal fetuses. VMAX in AO and PA decreased remarkably in the three abnormal groups. It was concluded that the kidneys were poorly perfused in the growth retarded fetuses with oligohydramnios, which may, in part, depend on the decreased stroke volume.

A case of trisomy 21 with holoprosencephaly: the fifth case.

A case of trisomy 21 with holoprosencephaly, the fifth case in the literature, is described. The patient also had extracephalic malformations frequently associated with Down syndrome. The possibility of a causal relationship between trisomy 21 and holoprosencephaly is discussed.

[Fetal therapy in utero by blockage of the umbilical blood flow of acardiac monster in twin pregnancy].

We studied a life-saving method for the unaffected twin in two recent patients who had acardiac monster in twin pregnancy. We succeeded in blocking the umbilical blood flow of the acardiac monster in utero as radical treatment for this abnormality in one of the patients. In case 1, tocolysis and amniocentesis with puncture to the cyst of the acardiac monster were performed to prevent premature labor. However, the patient underwent premature labor at 27 weeks of gestation, and the newborn died. In case 2, fetal treatment was given: A steel coil was placed in the umbilical cord close to the abdominal wall of the acardiac monster under ultrasonographic guidance at 23 weeks of gestation to block blood flow. As a result, no enlargement of the acardiac monster was observed, and the cardiac function of the unaffected fetus improved. At 38 weeks of gestation, the patient delivered a normal baby weighing 2,237g and an acardiac monster weighing 110g. There were no complications in either the mother or newborn. There has been no report describing blockage of the umbilical blood flow of an acardiac monster in utero. Our method is considered efficient and less risky to the mother and the unaffected twin.

Non-invasive continuous fetal transcutaneous pO2 and pCO2 monitoring during labor.

Continuous measurements of fetal scalp transcutaneous pO2 (tcpO2) and pCO2 (tcpCO2) monitoring were performed for 49 patients for the purpose of the evaluation of the fetal well-being. The equipment used was transcutaneous pO2/pCO2 monitor Micro Gas 7640 (KONTRON, Switzerland), which was inserted through the vagina and attached to the fetal head with a suction ring. Blood was taken from the umbilical vessels and pO2 and pCO2 values were analyzed. The correlation coefficient between pO2 in the umbilical artery and tcpO2 was 0.78 (p less than 0.01), and that between pCO2 in the umbilical artery and tcPCO2 was 0.79 (p less than 0.01). Relationships between the transcutaneous blood gas analysis and FHR patterns were discussed. Increase of pCO2 and decrease of tcpO2 was observed in the cases of variable deceleration, not observed in the case of early deceleration. FHR patterns don't always indicate fetal condition, for example in the case of fetal arrhythmia, especially fetal bradycardia. But tcpO2 monitoring of the fetus affected fetal arrhythmia during labor aids diagnosis of fetal distress by FHR patterns, especially in the case of fetuses affected by fetal bradycardia.

The prenatal determination of glucose-6-phosphatase activity by fetal liver biopsy.

Two fetuses at risk for glucose-6-phosphatase deficiency had in utero liver biopsies. Analysis of each showed this enzyme activity to be in the normal range and the pregnancies continued. Neither child has any clinical or metabolic evidence of glucose-6-phosphatase deficiency.

Ontogeny of fetal liver glucose-6-phosphatase activity.

A reliable and sensitive microassay for the measurement of liver glucose-6-phosphatase is described. Human fetal liver was assayed for glucose-6-phosphatase activity from 7.5 to 24 weeks of gestation and was found to have a mean activity of 2.11 nmol per min per mg of protein. This was approximately 30 per cent of the postnatal controls assayed by the same method, but there was no evidence of a change in activity during the gestational period examined. If fetal liver tissue can be reliably obtained, it may be possible to determine a deficiency of glucose-6-phosphatase in fetuses who are at risk.

[Fetal blood sampling by liver puncture].

Fetal blood sampling has been performed in several ways; placentacentesis, fetoscopy, or umbilical code puncture. The problems with these methods are technical difficulties and contamination of maternal blood or amniotic fluid. To solve these problems, we have tried fetal blood sampling by fetal liver puncture with a 21 approximately 23 gauge needle through the maternal abdomen under real time scan guidance. 10 patients underwent this procedure. They ranged from 18 weeks to 22 weeks of gestation at the time of sampling. The sampling procedures were done easily and the samples taken were shown to be pure fetal blood by red blood cell sizing. All the patients continued pregnancy after the examination and none of the pregnancies was influenced by the puncture. 7 patients have been delivered with neither sampling scars nor damage to liver function. This method provides pure fetal blood, the procedure is simple and, in our experience, no complications have occurred.

Abortifacient effect and uterine cervix-dilating action of 16, 16-dimethyl trans delta 2 PGE1 methyl ester (ONO 802) in the form of a vaginal suppository (a randomized, double-blind, controlled study in the second trimester of pregnancy).

A newly synthesized 16, 16-dimethyl trans delta 2 PGE1 methyl ester (ONO 802) was clinically applied in the form of a vaginal suppository for therapeutic abortion of second trimester pregnancies. Its effects were studied in a double-blind test comparing it with an inactive placebo suppository. The study was conducted at 12 Japanese university hospitals. The number of patients was 125 in total, i.e. 63 receiving ONO 802 vaginal suppository (containing 1.0 mg ONO 802) and 62 receiving inactive placebo suppository. ONO 802 was more effective than placebo with a success rate of 87% (complete (71%) and incomplete (16%) abortions). The onset of uterine contractions was observed in 154.3+/-18.1 min. and the onset of uterine bleeding in 323.6+/-41.0 min. The expulsion of the fetus and placenta was observed in 955.4+/-97.0 and 961.6+/-97.0 min., respectively. The cervix dilating effect of ONO 802 was observed in 63.5% of the patients at 3 hours after the start of administration. Nausea, vomiting, abdominal cramps, diarrhea and pyrexia were noticed. However, all these side effects were transient and mild, requiring no treatment. No abnormality was observed in the puerperal course, duration of uterine bleeding or onset of subsequent menstruation following the therapeutic abortion. Therefore, the present study demonstrated that ONO 802 vaginal suppository was an effective and valuable drug for therapeutic abortion of second trimester pregnancies.

[Effects of prostaglandin E1 analogue and prostaglandin F2 alpha on uterine contraction, uterine blood flow and fetal circulation in pregnant and nonpregnant rabbits (author's transl)].

Effects of PGF2 alpha and PGE1 analogue (ONO-802) on uterine contraction, uterine blood flow and fetal circulation were studied in pregnant and mature nonpregnant rabbits. Uterine and fetal blood flows were measured by photoplethysmography. Ten ng of PGs were injected into aorta abdominalis. In pregnant rabbits, 10 ng of PGF2 alpha caused a great increase in uterine contraction (i.e., intrauterine pressure), slight decrease in uterine blood flow and a transient fetal bradycardia, while 10 ng of ONO-802 caused a slight increase in uterine contraction (i.e., frequency), moderate decrease in uterine blood flow and a transient fetal bradycardia. Responses in nonpregnant rabbits to these two PGs were almost the same as those in pregnant rabbits. These results indicate the different effects of these two PGs on the uterine blood flow.

Effects of domperidone on serum prolactin levels in human beings.

Effects of domperidone, a dopaminergic antagonist, on serum prolactin levels were studied in 6 normal men and 6 normal cyclic women at the different phases of their menstrual cycles (i.e., the follicular, the preovulatory and the luteal phases). Domperidone (10 mg, i.v.) caused significant increases in serum prolactin in all cases within 15 min after the injection. The prolactin response was significantly (p < 0.01) higher in women than in men, and there was no significant difference in the prolactin responses among the three phases of the menstrual cycles. These results indicate that domperidone may be an effective stimulator of serum prolactin secretion in human beings.