Identification of a major carbohydrate capping group of the L-selectin ligand on high endothelial venules in human lymph nodes as 6-sulfo sialyl Lewis X.

We investigated the molecular species of sulfated sialyl Lewis X determinants, the putative L-selectin ligand, expressed on high endothelial venules (HEV) in human lymph nodes. Comparison of the reactivity pattern of HEV with the reactivity of the pure 6-sulfo, 6'-sulfo, or 6,6'-bissulfo sialyl Lewis X determinant with hitherto known anti-sialyl Lewis X antibodies strongly suggested 6-sulfo sialyl Lewis X to be the best candidate for the major sulfated sialyl Lewis X determinant on HEV, followed by 6,6'-bissulfo sialyl Lewis X, whereas 6'-sulfo sialyl Lewis X was unlikely. We newly generated monoclonal antibodies (mAbs) G152 and G72 directed against 6-sulfo sialyl Lewis X, which intensely labeled HEV in immunohistochemical examination and inhibited binding of recombinant L-selectin-IgG to HEV, suggesting that the determinant serves as a ligand for L-selectin. To test the concomitant expression of 6, 6'-bissulfo sialyl Lewis X, specific mAbs (G2706, G27011, G27037, and G27039) were generated, but all antibodies failed to react to HEV. Next, we established mAbs (AG97 and AG273) directed against 6-sulfo Lewis X, the asialo form of 6-sulfo sialyl Lewis X. The antibodies were not reactive to untreated HEV, but strongly reacted to sialidase-treated HEV. This indicated the predominance of the sialylated form of 6-sulfo sialyl Lewis X and minimal expression of its asialo form, corroborating that it was synthesized by fucosyltransferase VII, the isoenzyme that preferentially produces the sialylated form of the determinant.

Altered mRNA expression of specific molecular species of fucosyl- and sialyl-transferases in human colorectal cancer tissues.

Human colorectal cancers express various cancer-associated carbohydrate determinants such as Lewis Y or sialyl Lewis A, suggesting a considerable alteration in glycosyltransferase activities occurring upon malignant transformation. We investigated the mRNA amounts of fucosyltransferase (Fuc-T) and sialyltransferase (ST) isoenzymes, including Fuc-T III, IV, V, VI and VII and ST-3N, ST-30 and ST-4, in human colorectal cancer tissues by Northern blotting and RT-PCR. Regarding fucosyltransferases, mRNA of Fuc-T III and VI was not significantly altered, and only Fuc-T IV mRNA showed a moderate increase in cancer tissues when compared with adjacent non-malignant colonic epithelia taken from the same patient (273 +/- 96%; p < 0.001). The moderate increase of Fuc-T IV message may be related to an enhanced expression of Lewis Y in colon cancer tissues. In the ST isoenzymes, mRNA for ST-3N remained unchanged, whereas that for ST-4 decreased significantly in cancer tissues, to 32 +/- 29%, (p < 0.005). The most remarkable finding was that the message of ST-30 was prominently increased in cancer tissues compared with non-malignant colorectal mucosa. When further investigated by quantitative RT-PCR assays on a larger series of patients with colorectal cancers, the average increase in mRNA for ST-30 was 459 +/- 200% compared with that in adjacent non-malignant epithelium (significant at p < 0.0001). The increase of ST-30 message was more prominent in the cancer tissues strongly expressing sialyl Lewis A than in the cancer tissues expressing sialyl Lewis A only weakly or moderately (significant at p < 0.05). The marked increase in the message of ST-30 is suggested to be related to an enhanced expression of sialylated carbohydrate determinants in colon cancer tissues including sialyl Lewis A, since the enzyme exhibited a significant activity against the type 1 chain carbohydrate substrate and produced the precursors for sialyl Lewis A synthesis, when its cDNA was expressed in Cos-7 cells.

Effect of interleukin 2 on the production of progesterone and prostaglandin E2 in human fetal membranes and its consequences for preterm uterine contractions.

Our objective was to clarify the mechanism of uterine contraction induced in pregnant women by intrauterine bacterial infection. The concentration of interleukin 2 (IL-2) was measured in amniotic fluids that had been obtained by amniocentesis, transvaginal amniotomy or by transuterine amniocentesis performed at cesarean section in 50 pregnant women. The concentration of IL-2 in those cases with intrauterine infection was significantly higher than that of those without intrauterine infection at preterm. The same tendency was found at term. Scatchard analysis demonstrated the presence of an IL-2 receptor in the fetal membranes. We collected the fetal membranes aseptically for the measurement of progesterone and prostaglandin E2 by radioimmunoassay following incubation with various concentrations of interleukin 1 (IL-1) and IL-2 at 37 degrees C for 16 h. The production of progesterone was inhibited significantly by 10 pmol/l IL-2 but not by 10 pmol/l IL-1. The production of prostaglandin E2 was accelerated significantly by either IL-1 or IL-2 at a dose of 10 pmol/l. The inhibitory effect of IL-2 on the production of progesterone was unaffected by indomethacin, which inhibits the production of arachidonate cycloxygenase metabolites such as prostaglandin E2. Our present data suggest that the presence of intrauterine bacterial infection may stimulate the intrauterine production of IL-2, and that the stimulation of IL-2 and the reduction of progesterone caused by IL-2 may in part explain the mechanism of uterine contraction associated with intrauterine infection during pregnancy.

[Determination of intrauterine pressure using catheter-tip transducer inserted outside fetal membranes].

To determining intrauterine pressure outside fetal membranes, we used a catheter-tip transducer to study 20 women before the occurrence membrane rupture. Their mean age was 28.2 +/- 3.4 years and all women were in weeks 37 to 41 of pregnancy when studied. In the first stage of labor, the peak intrauterine pressure was 60.0 +/- 12.5 mmHg (mean +/- SD) when the external os was dilated 4 to 6 cm, 90.0 +/- 14.8 mmHg, at 7 to 8 cm dilation, and 80.0 +/- 11.5 mmHg at 9 cm or greater dilation. Each pressure wave lasted 45 to 55 sec. The highest baseline pressure (28.0 +/- 4.5 mmHg) was obtained when the subjects were sitting. A baseline pressure of 17.0 +/- 4.0 mmHg was obtained in the supine position, as well as a value of 21.0 +/- 3.5 mmHg in the recumbent position. There were no complications related to the catheter-tip transducer. Our findings indicate that this method is both accurate and reliable in determining the amounts of intrauterine pressure to which fetal membranes are subjected.

Enhanced proliferation of fetal rat hepatocytes in primary culture induced by ritodrine.

OBJECTIVE: Although ritodrine crosses the placenta, its direct effect on fetal cell proliferation has not been reported. We hypothesized that beta 2-adrenergic receptor stimulation could promote fetal liver growth. STUDY DESIGN: Ritodrine was added to serum- and hormone-free primary cultures of fetal, neonatal, or adult rat hepatocytes. We measured both tritiated thymidine incorporation into deoxyribonucleic acid and nucleus number. The effect of ritodrine on cell cycle was also analyzed with flow cytometry. RESULTS: Ritodrine enhanced the proliferation of fetal rat hepatocytes. Ritodrine remarkably stimulated deoxyribonucleic acid synthesis of fetal and neonatal but not adult hepatocytes. The effect was dose dependent and was antagonized by propranolol. Analysis of the nuclear deoxyribonucleic acid content derived from flow cytometry revealed that cells stimulated by ritodrine entered S phase. CONCLUSION: These results indicate that ritodrine may promote the proliferation of fetal hepatocytes through the stimulation of beta 2-adrenergic receptors, followed by induction of deoxyribonucleic acid synthesis.

Relationship between the changes in placental blood flow resistance assessed by Doppler technique and maternal serum placental aminopeptidases, which degrade vaso-active peptides, in pre-eclampsia.

Our study showed that there were statistically significant correlations between the systolic and diastolic ratio (S/D) of maternal uterine or umbilical artery and the levels of maternal serum aminopeptidase activities in pre-eclampsia. Kininase I was positively correlated with the S/D ratios, whereas placental leucine aminopeptidase (P-LAP) and aminopeptidase A were negatively correlated with the S/D ratios. It is known that the increased S/D ratios reflect the increased utero-placental blood flow resistance. Since our previous study showed that placental aminopeptidases degrade vasoactive peptides such as oxytocin, angiotensin and bradykinin, which the fetus actively produces, our present study suggests that the increased vascular resistance in feto-placental circulation in pre-eclampsia is partly controlled by changes in vaso-active peptides, via degradation by placental aminopeptidases.

The effect of nifedipine and dipyridamole on the Doppler blood flow waveforms of umbilical and uterine arteries in hypertensive pregnant women.

In a study of 45 hypertensive pregnant women, the systolic velocity/diastolic velocity ratio and pulsatility index of the umbilical and uterine arteries showed good correlation with the maternal blood pressure, and they appeared to provide a good parameter for the fetoplacental condition. Using the pulse Doppler method, we studied the effects of the antihypertensive agent nifedipine and of dipyridamole (an agent used to treat proteinuria) on the blood flow of the umbilical and uterine arteries in 16 hypertensive pregnant women. The results proved that both drugs caused a decrease in the vascular resistance of the umbilical artery and suggested that they increased the blood flow volume of this artery and were useful in the treatment of hypertension during pregnancy.

Induction by cortisol of aminopeptidases production from the human placenta in tissue culture.

Human placental aminopeptidase M and A and post-proline endopeptidase are known to act as degrading enzymes of bioactive peptides such as angiotensin II, oxytocin and endogenous opioids. We tested the effects of cortisol on the activities of human placental aminopeptidase A and M and post-proline endopeptidase using short-cultured placental tissues. From 34.5 nM to 3.45 microM of cortisol significantly increased the activities of 3 enzymes. Our present data suggest a possible important role of cortisol in the growth of human placenta via induction of placental aminopeptidases.

Rapid determination of fetal sex using amniotic fluid cells and the polymerase chain reaction.

We determined the sex of 50 fetuses by an amplification of the Y-chromosome specific fragment using the polymerase chain reaction (PCR). Amniotic fluid cells were collected by amniocentesis from pregnant women at 14 to 17 weeks of gestation. Total DNA was purified from cells in 1 ml of amniotic fluid. When only the expected 130 base pair X-chromosome specific fragment was detected, we identified the fetus as female, while when both the expected 170 base pair Y-chromosome specific and X-chromosome specific fragments were detected, we identified it as male. In all cases, identification was confirmed either by chromosome analysis or post partum.

Antenatal diagnosis of twin-twin transfusion syndrome by Doppler ultrasound.

We evaluated 31 pairs of twins: six with twin-twin transfusion syndrome, four discordant, and 21 concordant twins. The criterion for the retrospective diagnosis of discordancy was a difference in birth weight of 20% of the larger infant's weight. Using pulsed Doppler evaluation of the umbilical artery of each twin, the pulsatility index (PI) was used to evaluate the umbilical arterial waveforms. The differences in the PI from the twin-twin transfusion syndrome cases between 24-31 weeks significantly exceeded those of the cases without this syndrome (P less than .05). Seven cases had inter-twin differences in PI above 0.5, six of which had the twin-twin transfusion syndrome. The difference in PI seemed to predict the risk of twin-twin transfusion syndrome: The difference was high before the appearance of hydrops fetalis in those with this syndrome, whereas the difference in PI from discordant twin cases, except for those with the syndrome, was low.

Purification and properties of human placental aminopeptidase B.

Aminopeptidase B (EC 3.4.11.6; L-arginyl-beta-naphthylamidase) was purified 1,800-fold from human placental cytoplasm and characterized. The enzyme was subjected to ammonium sulfate fractionation and a series of chromatographies on DE-52, hydroxylapatite, Bio-gel A 0.5 m and L-arginine-Sepharose. The native molecular mass of the enzyme was estimated to be 220,000 by gel filtration. The molecular mass was estimated to be about 83,000 by SDS/PAGE in the absence of 2-mercaptoethanol, suggesting that the enzyme exists in a polymeric form. The isoelectric point of the enzyme was 5.4. The purified enzyme was most active at pH 7.2 with L-arginyl-beta-naphthylamide as substrate and the Km value for this enzyme was 0.3 mmol/l. Human placental aminopeptidase B was markedly activity by Cl-. Bestatin and arphamenin, low molecular weight peptides, showed appreciable inhibition of this enzyme. However, amastatin and puromycin did not inhibit the enzyme. Bacitracin markedly activated this enzyme.

Effects of low molecular weight peptides and divalent cations on degradation and binding of angiotensin II.

The effects of peptide inhibitors (bestatin and amastatin) and divalent cations (Ca2+ and Co2+) on the velocity of Asp1 liberation from angiotensin II (A-II) by human placental membrane fractions and binding of 125I A-II to human placental membranes were tested at 22 degrees C and 4 degrees C. Asp1 liberation was measured by high performance liquid chromatography. As expected, the degradation and binding of A-II were temperature sensitive, with both being at 4 degrees C than at 22 degrees C. While amastatin (10(-4) M) and bestatin 10(-6) M) significantly reduced the velocity of Asp1 liberation from A-II to about 45%, amastatin (10(-4) M) and bestatin (10(-4) M) increased 125I A-II binding to 125% and 130%, respectively. Ca2+ (10 mM) and Co2+ (10 mM) activated the velocity of Asp1 liberation from A-II to 140% and 120%, respectively at 22 degrees C. Ca2+ (10(-1) M) and Co2+ (10 mM) also enhanced 125I A-II binding about 130%. Previously we showed that the A-II degrading activity found in human placental membrane fractions is mainly due to aminopeptidases A and M. Since amastatin and bestatin are the specific inhibitors for aminopeptidases A and M, and since Ca2+ and Co2+ are the activators for aminopeptidase A and aminopeptidase M, respectively, it is conceivable that the enzymes regulate the levels of A-II and, therefore, that they may play an important role in the binding of A-II to human placental membrane fractions.

Increased serum kininase I activity in pregnancy complicated by pre-eclampsia.

Serum kininase I activity was measured in normal pregnancy and pre-eclampsia. The mean value in nonpregnant controls was 180 +/- 25 (SD) nmol/min/ml. Kininase I activity during normal pregnancy significantly increased after week 14, reaching the highest value (240 +/- 20 nmol/min/ml) at weeks 38 and 40. The kininase I activity in pregnancy complicated by severe pre-eclampsia was higher than that in normal pregnancy. The possible role played by elevated kininase I levels in pre-eclampsia was discussed.

A comparison of the binding of angiotensin II to human placenta between normal and severe pre-eclamptic pregnancy.

We compared the binding of angiotensin II (A-II) to human placenta between normal and severe pre-eclamptic pregnancy. Our data did not show any statistically significant difference between normal and pre-eclamptic pregnancy in both the equilibrium dissociation constant (Kd) and the total number of placental A-II receptors. Therefore, changes in the binding of A-II to placenta might not be involved in the possible cause of activation of the renin-angiotensin system in the feto-placental unit in severe preeclampsia.

Changes in the binding of angiotensin II to rat placental receptor by estrogen and progesterone.

The effects of estradiol and progesterone on the binding of rat placental angiotensin II receptors were examined. Sex steroid (progesterone estradiol plus progesterone) decreased the total number of rat placental angiotensin II receptors, while sex steroid (estradiol plus progesterone) increased angiotensin levels. Our present results suggest that sex steroids may play an important role in the control of the number of the angiotensin binding sites during pregnancy.

Sn-1,2-diacylglycerols and phorbol ester stimulate the production of progesterone from the human placenta.

Human term placental explants were used to investigate the possible role of phospholipid-sensitive and Ca2+ dependent protein kinase in the regulation of human placental progesterone production. Placental tissue was incubated with low density lipoprotein as a precursor of progesterone in the presence or the absence of phorbol 12-myristate-13-acetate, 1-oleoyl-2-acetyl-glycerol, and the calcium ionophore A23187. The rate of progesterone production by placental tissue was 21.7 +/- 4.6 ng. (mg wet wt)-1.(2 h)-1 (mean +/- SEM) with 500 mg low density lipoprotein/l (control). The rate of progesterone production was accelerated 2-fold by 1 nmol/l phorbol 12-myristate-13-acetate, 1.6-fold by 250 mumol/l 1-oleoyl-2-acetyl-glycerol and this increase was dose-related (25-250 mumol/l 1-oleoyl-2-acetyl-glycerol). A nonpromoting derivative, 4 alpha-phorbol-12,13-didecanoate had no effect. The phorbol 12-myristate-13-acetate or 1-oleoyl-2-acetyl-glycerol induced stimulation of progesterone production was not associated with a change in the intracellular cAMP level. Addition of 10 mumol/l A23187 further increased progesterone production with 125 mumol/l 1-oleoyl-2-acetyl-glycerol. The rate of progesterone production was accelerated 1.6-fold by 125 mumol/l 1-oleoyl-2-acetyl-glycerol and 10 mumol/l A23187 as compared with control. The effects of the phorbol ester and the diacyl glycerol were completely blocked by the addition of the protein synthesis inhibitor cycloheximide. We conclude that these phorbol regents are able to stimulate human placental progesterone production. The possible roles of intracellular Ca2+ and protein kinase C in placental steroidogenesis are discussed.

Effects of bestatin on intrauterine growth of rat fetuses.

Pregnant Wistar rats were injected with bestatin, a specific inhibitor of aminopeptidase M. Placental aminopeptidase M activity was inhibited by injection of bestatin, and fetal body weight was statistically lower than that in the saline-injected or control group. Our present data suggest that placental aminopeptidase M plays an important role in fetal growth.

Increased sensitivity to bradykinin in pregnant rats.

We tested the effects of late pregnancy on the depressor response to bradykinin (100, 200 and 400ng) in rats. All experiments were performed under anesthesia by intraperitoneal injection of pentobarbital (50 mg/kg). Catheters were connected to the arterial and venous lines for blood pressure recording and administration of drugs. Late pregnancy (19- to 21-day) showed a hypersensitivity of the depressor response to bradykinin. The effects of captopril, Kininase II inhibitor, on the depressor response to bradykinin were examined in rats. Since captopril (50, 100 and 200 micrograms) notably increased the depressor response to bradykinin both in nonpregnant and 19- to 21-day pregnant rats, kininase II acts as a bradykinin metabolizing enzyme in vivo. Captopril (50, 100, 200 micrograms), however, resulted in the augmented parallel increases of depressor response to bradykinin (100ng) in both nonpregnant and pregnant rats. Our data may suggest that kininase II is not involved in the hypersensitivity to bradykinin in late pregnancy in rats.