Umbilical vein thrombosis as a possible cause of perinatal morbidity or mortality: report of two cases.

Significant occlusion of the lumen of the umbilical vein by thrombus was observed in 2 patients with unexplained intrauterine fetal death (IUFD)/fetal distress. Although a normal non-stress testing result was obtained 7 days prior to IUFD in one patient, IUFD was noted during regular antenatal care at 39 weeks of gestation; intrapartum abrupt onset of deceleration in fetal heart rate pattern was observed at 40 weeks of gestation, lasting 14 min until vacuum extractor-assisted delivery in the other patient. Umbilical vein thrombosis was considered contributory to IUFD and the abrupt deterioration in fetal heart rate pattern in these 2 patients. Histological examination of the umbilical cord is thus important in unexplained fetal death/fetal distress.

Effect of zinc on carp (Cyprinus carpio L.) erythrocytes.

The effect of zinc exposure on some properties of the carp erythrocyte membrane was studied in vitro. Red blood cells plasma membranes were separated from other cellular membranes using a combination of differential and density gradient centrifugation. The purity of obtained plasma membrane preparations was determined by measuring the activity of the marker enzymes. Electrophoretic patterns of the main erythrocyte membrane proteins excluded their degradation during the isolation and purification procedure. Carp erythrocyte membranes, obtained from cells previously incubated with increasing ZnSO4 concentrations, were used to elucidate the effect of zinc ions on their physical and biochemical properties. Using fluorescent probes: 12-AS and TMA-DPH, we found that zinc ions reduced the fluidity of the lipid bilayer, both in the middle and near the aqueous interface. Moreover, it was observed that zinc had no significant influence neither on the Na,K-ATPase activity nor on the thiol groups content in the erythrocyte membrane. We also detected that incubation of erythrocytes with zinc lead to the marked decrease of hemolytic resistance of the cells. Our studies demonstrate that zinc at higher concentrations may be toxic to carp erythrocytes causing changes in the membrane fluidity and hemolytic resistance.

Zinc-induced damage to carp (Cyprinus carpio L.) erythrocytes in vitro.

Fish erythrocytes were used to elucidate the effect of zinc ions on the cell antioxidant defence system. It was detected that an increase of the Zn2+ concentration (0.01-1 mM) leads to a marked decrease (p < 0.05) in the catalase and the glutathione peroxidase activities. We observed a loss of 14-39% activity of glutathione peroxidase, and 16-20% diminution for catalase. No significant changes were found in case of the superoxide dismutase. Incubation of red blood cells with zinc brought about a decrease of the erythrocyte thiol group content. Treatment of carp erythrocytes with zinc ions also resulted in enhanced hemolysis and in the induction of significant (p < 0.001) changes in the intracellular glucose level. The increase of glucose concentration in the erythrocytes was correlated with increased concentration of metal in the incubation medium. It was proposed that Zn could affect transport systems across the red blood cells and therefore increased the permeability of the membranes to small molecules (e.g. hexose), and led to hemolysis. Zinc ions could act as a potential cell toxicant, leading to disturbances in functions of the antioxidant defence system and to alterations in the erythrocyte membrane properties.

Application of a licorice root specific protein to a general method for the assay of licorice root components in traditional Chinese medicines.

A new fuzzy immunoassay method generally applied to ten Glycyrrhizae Radix (GR) preparations of four different botanical origins was studied. Four kinds of antisera were elicited in rabbits immunized with GRs of different botanical origins. The presence of the characteristic GR protein (GRP) was shown using Western blot analyses and selected antibody enzyme immunoassay (SAEIA) methods. A GRP was isolated from one of the GR specimens which was selected using SAEIA methods. The isolated GRP was heated to reduce its binding activity to an anti-GR serum. A new fuzzy SAEIA method generally applicable for assay of the extract of the ten GR specimens was developed using heat-treated GRP as the solid-phase antigen. The fuzzy SAEIA method was successfully applied for the detection and quantitative analysis of the GR component contained in traditional Chinese medicines.

A general enzyme immunoassay for the licorice root component contained in traditional Chinese medicines.

The development and application of a new enzyme immunoassay for general assay of the Glycyrrhizae radix (GR) component in Chinese traditional medicines is described. Three commercial GR-based medicines, Tohoku kanzo (GRTK), Seihoku Kanzo (GRSEK) and Sinkyo kanzo (GRSK) were used as GR specimens. Anti-GRSK serum was elicited from rabbits immunized with GRSK fragments. The presence of common proteins as specific antigens of GR was first established by Western blot analysis of extracts of GRSK, GRSEK or GRTK using anti-GRSK. The specific antigens were applied to develop an ELISA for the assay of GR extract. Anti-GRSK was put in competition with a sample or standard GR extract and immobilized GRSK components in microtiter plate wells. The proportion of antibody binding to the solid-phase GRSK component was detected using an enzyme-labeled second antibody. The ELISA method was specific to GRSK extract and showed low sensitivity for the assay of GRTK extract. The technique of selected antibody enzyme immunoassay (SAEIA) was applied to develop a sensitive general assay method. Solid-phase GRTK extract, rather than immobilized GRSK extract, was used in the SAEIA. The SAEIA possessed the same quantitative working range of between 1 and 100 micrograms/ml for the assay of each extract of GRTK, GRSK and GRSEK. The SAEIA was successful in the detection and quantitative measurement of GR component contents in Chinese traditional medicines.

A new method for the detection and quantitative measurement of the contents of trichosanthes root component composing Chinese traditional medicines.

A new method was developed to estimate the content of Trichosanthes root (TR) component in two Chinese traditional medicines. Characteristic antigens of TR were separated from TR extract using rabbit antiserum specific for TR, a dried root tuber of Trichosanthes kirilowii. Two selected antibody enzyme immunoassay (SAEIA) methods, the SAEIA A for assay of TR extract and the SAEIA B for assay of karasurin A were used as detection methods for the separation of TR antigens. Using several column chromatographies, two kinds of TR antigens, a protein component and a glycan component, were separated from TR extract. A SAEIA C method for assay of TR glycan component was developed using biotinylated second antibody and peroxidase-labeled avidin as the detection method. The SAEIA C was also found applicable for specific assay of TR extract as well as for the contents of TR component present in two Chinese traditional medicines, prescriptions of which contained TR. Content of trichosantin, an abortifacient protein component of T. kirilowii, in both the medicines were also measured by applying the SAEIA B for assay of karasurin A. Little trichosantin was found in either medicine and the reason for this was determined.

JunD mutants with spontaneously acquired transforming potential have enhanced transactivating activity in combination with Fra-2.

Although a replication-competent retrovirus that carries junD has no transforming activity in chicken embryo fibroblasts, we have isolated mutant viruses that have spontaneously acquired transforming activity. The molecularly cloned junD genes of three such mutant viruses (T1, T2, and T3) were shown to be responsible for the cellular transformation. DNA sequence analysis indicated that a specific polynucleotide in the junD sequence was tandemly multiplied three times of five times in T1 and T2, respectively. The repeated polynucleotide encodes 16 amino acid residues that are located in a highly conserved region among Jun family proteins. The junD mutation in T3 involved an inversion, a translocation, and nucleotide substitutions that caused drastic amino acid exchanges in another well-conserved region among Jun family proteins. The transcriptional activity of these mutants was analyzed by means of transient expression experiments in F9 cells using a reporter gene containing a single AP-1 binding site. Compared with the wild-type JunD, none of them showed enhanced transactivating activity in the forms of homodimers or of heterodimers with c-Fos or Fra-1. However, they did exhibit much higher transactivating activity than the wild type when they formed heterodimers with Fra-2, indicating that the mutated regions function as transactivation domains in a partner-specific manner. Since we have previously reported that there is a basal level of Fra-2 expression in chicken embryo fibroblasts, the results may indicate that protein complexes between JunD mutants and Fra-2 play a crucial role in the cellular transforming activity.

Escape from redox regulation enhances the transforming activity of Fos.

Fos and Jun form dimeric complexes that bind to DNA sequences containing activator protein 1 (AP-1) sites and regulate gene expression. The in vitro DNA-binding activity of these proteins is sensitive to reduction-oxidation (redox). Reduction of a single conserved cysteine residue, located in the DNA-binding domain, either by reducing agents or by a nuclear redox factor (Ref-1), is required for AP-1 DNA-binding activity. Replacing the critical cysteine with serine results in a protein that can bind to DNA in vitro even under oxidizing conditions. To determine whether redox control affects the function of Fos in vivo, we have constructed, and compared the properties of, retroviral vectors expressing either a truncated Fos protein (F118-211) or a truncated Fos protein in which the critical cysteine was replaced by serine (FC154S). In infected chicken embryo fibroblasts (CEFs), both vectors expressed similar levels of Fos protein, which formed heterodimers with Jun at equivalent efficiencies. However, extracts from cells expressing FC154S exhibited a threefold increase in AP-1 DNA-binding activity compared with cells expressing F118-211. Furthermore, this enhanced binding activity was resistant to treatment with the oxidizing agent diamide. Infection of CEFs by virus expressing FC154S resulted in increased numbers of transformed colonies and an increase in colony size compared with those obtained following infection by virus expressing Fos 118-211. These results suggest that redox regulation may limit the total level of functional Fos-Jun complexes in vivo and that escape from this control enhances transforming activity.

A new enzyme immunoassay for a solid Chinese crude drug, pinellia tuber.

A new immunoassay for a solid Chinese crude drug was studied. An antiserum specific for Pinellia tuber was elicited in two rabbits. Using the antiserum and powdered Pinellia tuber-coated microtiter plate as the immunological reagents, and beta-D-galactosidase-labeled goat anti-rabbit immunoglobulin G (IgG) as the tracer, a new enzyme immunoassay for a solid Pinellia tuber with a working range between 0.1 and 1000 micrograms/ml was developed. The assay was specific for a solid Pinellia tuber and showed low cross-reaction values on other Chinese crude drugs and the extract of Pinellia tuber. The specificity of the assay was compared with the selected antibody enzyme immunoassay (SAEIA) for the extract of Pinellia tuber recently developed. Both methods utilized the same immunological reagents such as the serum and the enzyme-labeled goat anti-rabbit IgG, and the only difference between them was the solid-phase antigen used. The assay results of several antigens determined by them were quite different, showing that selective measurements of different antigens, either solid or the extract of Pinellia tuber, were possible using the same antiserum, when the tracing reaction in the immunoassay was adequately selected.

Composition of cholesterol esters in blood plasma of mouse, rat and rabbit.

Mixtures of cholesterol estes were extracted from the blood plasma of mouse (C3H/He, C57BL/6J and Jcl : ICR), rat (Wistar) and rabbit (JW-NIBS/Rabiton) and purified by thin-layer chromatography. Fatty acid moieties of the esters were methylated with BF3-methanol and analyzed quantitatively by gas chromatography. Although linoleate was most abundant (over 30% of the total esters) and myristate was found only in traces, the composition of the esters varied markedly among the species. Mice had 15.3 to 17.4% of arachidonate, but only a trace of linolenate and no stearate. In rats, arachidonate and linoleate were found in almost equal quantities, but stearate, linolenate and docosahexaenoate were present in only trace amounts. Rabbits had more palmitate and oleate and less arachidonate and eicosapentaenoate than the other two animals, but no docosahexaenoate.

Hepatic 7-alkoxycoumarin O-dealkylase in mice: induction by beta-naphthoflavone of hepatic enzyme activity.

Hepatic 7-alkoxycoumarin O-dealkylation activities in control and beta-naphthoflavone-pretreated mice were determined. The O-demethylation and O-deethylation activities of 7-alkoxycoumarin in control mice were almost the same values, while the O-depropylation activity was lower than those of the other reactions. The O-dealkylase activity varied markedly among the 18 strains of mice surveyed, and strain-dependent differences in the cytochrome P-450 content were also detected among the strains. beta-Naphthoflavone induced O-dealkylation activity, especially O-deethylation and O-depropylation activities, only in ddY, DS and its substrains (A2-3, A3-1, C1-2 and Nh/+). C3H/He and C57BL/6J strains, but not in DBA/2, BALB/c and KYF/2 strains. The former strains of mice are thus classified as "responsive strains" to beta-naphthoflavone and the latter as "non-responsive strains". The O-dealkylase activity in other strains of mice were not clear in responsiveness to beta-naphthoflavone. The hepatic cytochrome P-450 content in responsive strains also increased upon pretreatment of the animals with beta-haphthoflavone. The results indicate marked strain differences in basal and beta-naphthoflavone-induced activity of hepatic 7-alkoxycoumarin O-dealkylase in mice.

Time course of biochemical and histological alterations following a single feeding of carbon tetrachloride to mice.

Carbon tetrachloride (0.02, 0.5 or 1.5 ml/kg) was given through a stomach tube to female C57BL/6J mice. Liver weights, the quantities of nine liver (protein, free glucose, glycogen, triglyceride, phospholipid, free and esterified cholesterol, DNA and RNA) and three blood components (glucose and free and esterified cholesterol), and SGOT activities were measured at 15, 24, 39, 48, 63, 87, 159 or 327 hr after carbon tetrachloride feeding. Livers were examined histologically. Chemical components of the liver and blood changed generally in a dose-related manner, but more slowly in the larger-dose groups. The biochemical alterations were reflected well in the histological findings. Degenerative and regenerative findings occurred later in the larger-dose animals. Histological changes appeared first in the midzones in the 0.5 and 1.5 ml/kg groups.