Isolation, characterization and synthesis of alpha-foetoprotein from neonatal-rat skin.

Monospecific anti-[rat alpha-foetoprotein(alpha-FP)] immunoglobulin G was coupled to CNBr-activated Sepharose-4B (4.5 mg/ml packed volume of gel) to yield an adsorbent. The immunoaffinity column was used to isolate alpha-FP from neonatal-rat skin. Purified skin alpha-FP was found to be immunologically and electrophoretically similar to serum alpha-FP. It yielded a single band with mol.wt. 68000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, on polyacrylamide-gel electrophoresis under non-denaturing conditions, the alpha-FP displayed slow- and fast-moving variants similar to those observed in serum alpha-FP. A Scatchard plot of oestradiol binding to the alpha-FP yielded an association constant of 2.5 X 10(9)M-1 by dextran-coated-charcoal and 0.75 X 10(8)M-1 by Sephadex-gel-filtration procedures respectively. Skin explants from newborn rats were found to incorporate [14C]leucine into immunoprecipitable intracellular alpha-FP. Cycloheximide inhibited the synthesis of alpha-FP in skin explant culture. Our results indicate that newborn-rat skin contains alpha-FP that is similar to serum alpha-FP and which may arise in neonatal-rat skin as a result of synthesis in situ.     

Development of the orbicularis oris muscle in normal and cleft lip and palate human fetuses using three-dimensional computer reconstruction

As part of an ongoing study of cleft lip and palate fetal morphology, normal and dysmorphic development of the human fetal orbicularis oris muscle was studied in a cross-sectional sample of 29 human fetuses (20 "normal" and 9 cleft lip and palate) ranging in age from 8 to 21 postmenstrual weeks. The specimens were embedded in celloidin and sectioned at 20 microns, and every tenth section was stained with hematoxylin and eosin. A computer reconstruction technique was applied to produce three-dimensional representations of the orbicularis oris muscle. The orbicularis oris muscle in the normal fetal sample with discernible lip fibers (N = 15) increased symmetrically in both fiber density and complexity from 12 to 21 weeks. Metrically, muscle volume and thickness growth curves were consistent with qualitative observations. In contrast, the unilateral cleft lip and palate fetal specimens with discernible lip fibers (N = 3) exhibited a 3.5-week delay in overall muscle development, asymmetrical fiber distribution, and abnormal fiber insertions. However, quantitatively, no significant (p greater than 0.05) differences were noted in orbicularis oris muscle thickness or volume between the normal and cleft lip and palate fetal specimens through 21 weeks. Findings suggest that orbicularis muscle deficiency, noted clinically in cleft lip and palate neonates, may be a result of perinatal functional dysmorphogenesis rather than congenital mesenchymal reduction or deficiency.     

Mouse alpha 1-fetoprotein and albumin. A comparison of their binding properties with estrogen and fatty acid ligands

The binding of estradiol-17 beta (E2), diethylstilbestrol (DES), and polyene fatty acids, in particular arachidonate (C20:4), to alpha 1-fetoprotein (alpha-FP) and albumin purified from mouse embryo sera was studied using equilibrium dialysis and electrophoretic techniques. E2, arachidonate, and DES all bind to alpha-FP, but with decreasing strength. E2 is a high affinity, low capacity ligand (Ka approximately 0.8 X 10(8) M-1 and approximately 0.3 sites/mol of alpha-FP at 25 degrees C); arachidonate is a weaker ligand disposing of more sites (Ka approximately 0.3 X 10(7) M-1 and 4-5 sites/mol of alpha-FP); the binding of DES is of comparatively low affinity and capacity (Ka approximately 0.2 X 10(7) M-1 and n approximately 0.7/mol of alpha-FP). In spite of different structures and equilibrium parameters, E2, DES, and arachidonate are able to compete with each other for binding to the fetoprotein. The C22:4 and C22:6 fatty acids are also efficient concentration-dependent inhibitors of E2 or DES binding. Albumin binds the fatty acids and DES, but equilibrium parameters are different from those of alpha-FP. In particular, arachidonate is a better ligand for albumin, where it interacts with at least two classes of apparent sites (Ka1 approximately 0.3 X 10(8) M-1 and n1 approximately 1; Ka2 approximately 0.2 X 10(7) M-1 and n2 approximately 30). In contrast to alpha-FP, albumin virtually does not bind E2. Also, no competition could be demonstrated between DES and fatty acid ligands for binding to albumin. None of the studied interactions, with either albumin or alpha-FP, was modified even by high doses of bilirubin. The possible functions of the various binding activities present in fetal sera in the process of growth are discussed.     

Participation of the lone tryptophan residue of rat alpha-foetoprotein in its drug-binding sites. Comparison with rat serum albumin.

The participation in drug binding of the lone tryptophan residue of rat alpha-foetoprotein (alpha-FP) and serum albumin, the two main transport proteins of foetal serum, has been studied by two different techniques. Firstly, the effect on phenylbutazone and warfarin binding of the chemical derivatization of the lone tryptophan residue of both proteins by 2-nitrophenylsulphonyl chloride (NPS) was studied. Secondly, the effect of phenylbutazone binding on the intrinsic fluorescence of the tryptophan residue of rat alpha-FP and albumin was investigated. The specific modification of the proteins by NPS did not affect the binding of warfarin by rat alpha-FP and albumin, but greatly decreased the affinity of the high-affinity sites of rat alpha-FP for phenylbutazone, though the numbers of these sites were not significantly changed. However, for albumin a similar decrease in the affinity constant appeared to be due to the reaction conditions. The spectrofluorimetric studies showed that the lone tryptophan residue of alpha-FP and albumin was quenched by phenylbutazone binding, and the quenching paralleled the fractional saturation of the high-affinity site only in the case of albumin. The effect of phenylbutazone binding on the intrinsic fluorescence of rat alpha-FP indicated that the lone tryptophan residue of this foetal protein is not in the same molecular environment as that of albumin, not participating directly in the high-affinity site for phenylbutazone, and the effect may be via some induced conformational change in rat alpha-FP. These results also confirm our previous suggestion that the high-affinity sites for phenylbutazone and warfarin are different on the rat alpha-FP molecule. The results seem to indicate that this is also the case for albumin, but confirmation is necessary.     

Dynamics of alpha-fetoprotein concentration in mice of different genotypes during the neonatal period]

A comparative assay of the alpha-fetoprotein (alpha-FP) level in mice of various genotypes (CBA, C3H, C57BL/Se/Sn, BALB/c, CC57W, AKR and nude--nu/nu) was conducted in the course of 3 weeks of postnatal period. The concentration of alpha-FP reached the following levels: the first day 2(-10)-2(-9); the 5th day 2(-8); the 8th day 2(-7); the 15th day 2(-4); on the 22nd day the level was zero. Nude mice which showed the alpha-FP concentration of 2(-2) on the 15th day were an exception. A conclusion was drawn that the alpha-FP synthesis was based not on the athymia of nude mice per se, but upon other unknown factors.     

Gastrin in fetal and neonatal pigs

1. The concentration and molecular profile of gastrin were examined in plasma and tissue extracts of fetal and neonatal pigs from 93 days gestation up to 12 weeks of age and also in the fetal gastric contents. 2. Gastrin was present in the gastrointestinal tract and plasma of fetal pigs at 93 days gestation. The concentration in both plasma and antral extracts increased progressively up to birth and continued to rise postnatally, reaching a peak at about 3 weeks of age in plasma and 6 weeks in the antrum. 3. In blood the major molecular form of gastrin was G34 (up to 80%), while in the antrum the major form was G17 (66-91%). The percentage of G34 in the antrum was highest in later gestation (21%), and reached adult proportion by 8 weeks of age (4%). 4. A considerable amount of gastrin, chiefly G17, was detected in the fetal gastric contents. Synthetic human G17 was stable in fetal gastric contents when incubated at 37 degrees C for 60 min, although, when incubated with gastric contents from a sow, it disappeared within 5 min. 5. It is suggested that the presence of gastrin in fetal gastric contents may be important in stimulation of fetal gut development.     

High Resolution Size Analysis of Fetal DNA in the Urine of Pregnant Women by Paired-End Massively Parallel Sequencing

Background

Fetal DNA in maternal urine, if present, would be a valuable source of fetal genetic material for noninvasive prenatal diagnosis. However, the existence of fetal DNA in maternal urine has remained controversial. The issue is due to the lack of appropriate technology to robustly detect the potentially highly degraded fetal DNA in maternal urine.

Methodology

We have used massively parallel paired-end sequencing to investigate cell-free DNA molecules in maternal urine. Catheterized urine samples were collected from seven pregnant women during the third trimester of pregnancies. We detected fetal DNA by identifying sequenced reads that contained fetal-specific alleles of the single nucleotide polymorphisms. The sizes of individual urinary DNA fragments were deduced from the alignment positions of the paired reads. We measured the fractional fetal DNA concentration as well as the size distributions of fetal and maternal DNA in maternal urine.

Principal Findings

Cell-free fetal DNA was detected in five of the seven maternal urine samples, with the fractional fetal DNA concentrations ranged from 1.92% to 4.73%. Fetal DNA became undetectable in maternal urine after delivery. The total urinary cell-free DNA molecules were less intact when compared with plasma DNA. Urinary fetal DNA fragments were very short, and the most dominant fetal sequences were between 29 bp and 45 bp in length.

Conclusions

With the use of massively parallel sequencing, we have confirmed the existence of transrenal fetal DNA in maternal urine, and have shown that urinary fetal DNA was heavily degraded.     

Developmental change in the position of the fetal human larynx

The position of the mammalian larynx has been shown to be an important determinant in breathing, swallowing, and vocalizing patterns. While the growth of the adult human larynx has been studied extensively, detailed examination of fetal development has not been undertaken. Thus, crucial developmental change in the fetal period and the effects of this change on normal maturation are still unclear. This study has examined the development of the larynx and its topography during the fetal period. Thirty specimens were preserved in 10% buffered formaldehyde solution for a period of 6 weeks, after which mid-sagittal sections were performed. Fetal ages were calculated from femur diaphyseal lengths and ranged from 15 to 29 weeks. Direct measurements were taken to determine the growth and position of the larynx and trachea relative to the vertebral column and soft palate. Results show that the upper and lower levels of the larynx correspond to the basiocciput and the lower border of the third to upper border of the fourth cervical vertebrae, respectively. The epiglottic cartilage was present at 15 weeks. By 21 weeks, the epiglottis was well developed and in close palatal apposition. At 23 to 25 weeks, the epiglottis and soft palate were found to be in full contact. The acquisition of this contact may be related to fetal respiratory viability.     

Changes in the rhythms of the alpha-fetoprotein content of the blood serum of mice with hepatoma 22A and of tumor cell proliferation depending on the time of cyclophosphane administration

A study was made of the content of alpha-fetoprotein (alpha-FP) in the blood serum and of proliferative processes in hepatoma 22A-bearing mice after injection of cyclophosphamide in a dose of 150 mg/kg at varying time of 4-day tumor growth. The data obtained indicate that the time of appearance of alpha-FP in the blood, the pattern of rhythmic fluctuations in protein content and in the number of DNA-synthesizing and mitotic cells of the tumor depend on the time of the cytostatic injection during the day. Besides, cyclophosphamide exerts a more powerful inhibitory effect on the content of alpha-FP in the blood serum and less powerful on the proliferative processes in hepatoma 22A.     

24-hour rhythms in oxidative stress during hepatocarcinogenesis in rats: effect of melatonin or alpha-ketoglutarate

To compare the effects of alpha-ketoglutarate (alpha-KG) and melatonin on 24-h rhythmicity of oxidative stress in N-nitrosodiethylamine (NDEA)-injected Wistar male rats, melatonin (5 mg/kg i.p.) or alpha-KG (2 g/kg through an intragastric tube) was given daily for 20 weeks. In blood collected at 6 time points during a 24-h period, serum activity of aspartate transaminase (AST) and alanine transaminase (ALT) and the levels of alpha-fetoprotein (alpha-FP) were measured as markers of liver function. To assess lipid peroxidation and the antioxidant status, plasma levels of thiobarbituric acid reactive substances (TBARS) and of reduced glutathione (GSH) were measured, together with the activity of erythrocyte superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST). NDEA augmented mesor and amplitude of rhythms in AST and ALT activity and plasma alpha-FP levels and mesor values of plasma TBARS, while decreasing mesor values of plasma GSH and erythrocyte SOD, CAT, GPx and GST. Acrophases were delayed by NDEA in all cases except for alpha-FP rhythm, which became phase-advanced. Co-administration of melatonin or alpha-KG partially counteracted the effects of NDEA. Melatonin decreased mesor of plasma TBARS and augmented mesor of SOD activity. The results indicate that melatonin and alpha-KG are effective in protecting from NDEA-induced perturbation of 24-h rhythms in oxidative stress. Melatonin augmented antioxidant defense in rats.     

Ring chromosome 21 in the mother and 21/21 translocation in the fetus: karyotype: 45,XX,-21,-21,+t(21;21)(p11;q11)

In 1984 we reported a ring chromosome 21 in a normal woman with recurrent fetal wastage (Kleczkowska and Fryns, 1984). A 46,XY normal fetal karyotype was found after prenatal diagnosis at 14 1/2 weeks in a third pregnancy of this woman. In the present paper we report the prenatal diagnosis of a 21/21 translocation in a female fetus from her fourth pregnancy.     

Maternal levels of pregnancy-specific β1 (SP-1) are elevated in pregnancies affected by Down's syndrome

Summary Concentrations of pregnancy-specific ₁-glycoprotein (SP-1) were measured in maternal blood and amniotic fluid of patients with a trisomic fetus and compared with that of a cytogenetically normal fetus at weeks 16–19 of pregnancy. The SP-1 concentrations were significantly elevated in the sera of women with a Down's syndrome fetus, whereas amniotic fluid levels were only slightly increased. It is suggested that high levels of maternal SP-1 in the second trimester of pregnancy may be a valuable indicator in the prenatal detection of fetal trisomy 21.     

Induction of alpha-fetoprotein synthesis in nonproliferating hepatocytes

The localization of alpha-fetoprotein (alpha-FP) and the proliferative activity of hepatocytes were studied after an insignificant liver damage. On the 3rd--6th day after the operation alpha-FP was detectable in hepatocytes localized on the boundary with the zone of damage. The most intensely stained cells were found adjacent to some vessels lying near this zone. The overwhelming majority of alpha-FP-containing hepatocytes did not incorporate 3H-thymidine. The data obtained allow a conclusion that the synthesis of alpha-FP is not related to cell proliferation.     

Expression of calcium-binding protein regucalcin mRNA in fetal rat liver is stimulated by calcium administration

The expression of hepatic calcium-binding protein regucalcin mRNA in fetal rats was investigated. The alteration in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb with complete open reading frame). Hepatic regucalcin mRNA levels were progressively increased with fetal development; the mRNA was clearly expressed at 15 and 21 days of pregnancy but only slightly at the 8 days. Meanwhile, -actin mRNA levels in the fetal liver were remarkable at 8 and 15 days of pregnancy. The fetal liver regucalcin mRNA levels at 15 days of pregnancy were significantly decreased by overnight-fasting of maternal rats. The oral administration of calcium chloride (50 mg Ca/100 g body weight) to maternal rats at 15 days of pregnancy caused a remarkable elevation (about 2 fold) of regucalcin mRNA levels in the fetal liver; this increase was seen 60 and 180 min after the calcium administration. After birth, regucalcin mRNA was increasingly expressed in the livers of newborn and weanling rats, while hepatic -actin mRNA expression was not appreciably altered with increasing ages. These findings demonstrate that the expression of hepatic regucalcin mRNA is increased with fetal development, and that the gene expression may be stimulated by the ingestion of dietary calcium.     

Abnormal cerebral cortical convolutions in an XYY fetus

Summary Because of maternal disease a pregnancy was electively terminated at 21 weeks of fetal gestation. At autopsy the fetus showed an abnormal pattern of cerebral cortical convolutions involving the frontal lobes. Karyotypic analysis revealed an XYY sex chromosome constitution. This observation, if confirmed in other XYY cases, raises the possibility that a developmental abnormality of the cerebral cortex may be involved in the higher-than-normal risk for behavioral disability exhibited by men with this genotype.Supported in part by Grants HD-05615 and HD-04612 from The National Institute of Child Health and Human Development.     

Expression of laminin subunits in human fetal skeletal muscle

Summary The laminin variant of adult skeletal muscle fibres and Schwann cells is known as merosin, and is composed of M-B1-B2 chains. Blood vessels and immature fibres express the A chain in association with B1 or S, and B2. The importance of merosin has recently been shown by its absence in one form of congenital muscular dystrophy and in the mutantdy/dy mouse, and by its partial deficiency in Fukuyama congenital muscular dystrophy. We have examined the immunocytochemical localization of the M, A, B1 and B2 laminin chains in human fetal muscle from 7 to 40 weeks' gestation to ascertain their developmental expression. The B1 and B2 chains were detected on muscle fibres at 7 weeks, but only traces of the A or M chain were seen. By 21 weeks maximal levels of all four subunits were observed on all fibres. This suggests that the basement membrane is still being assembled until this stage of development. Expression of the A chain on muscle fibres was not reduced until 34 weeks and low levels persisted at birth. The concomitant expression of the M and A chains at early stages may indicate a laminin variant, in addition to merosin, that is highly expressed in fetal muscle. Merosin was seen in intramuscular nerves at 11 weeks. B1 and B2 subunits were detected in blood vessels from 7 weeks' gestation and the A chain from 11 weeks. The capillary network, however, is not fully established in fetal muscle. Merosin is therefore detected early during human fetal muscle development, and this should be taken into account when assessing aborted fetuses at risk for congenital muscular dystrophy.     

Urethral and urachal urine output to the amniotic and allantoic sacs in fetal sheep

A chronic fetal sheep preparation was developed to measure, without interruption in utero, urethral and urachal urine output to the amniotic and allantoic sacs, respectively. Fetal urethral, urachal and total urine output was measured during a 5 day post-operative period, in late gestation. Total fetal urine output increased from day 1 to a volume of 1216 +/- 115 ml/day (SEM) on day 5 post-operative. Urachal urine output increased significantly from 12 ml/day on day 1 to 467 ml/day on day 5 (P less than 0.05). Fetal arterial blood gases, pH and immunoreactive ACTH, cortisol and immunoreactive arginine vasopressin concentrations were stable throughout the 5-day recovery period. Fetal urachal urine output to the allantoic cavity and total fetal urine output appears to require 4-5 days to stabilize post-operatively. Fetal urine is a major source of amniotic and allantoic fluid in late gestation and the volume of these sacs may be influenced, in part, by the distribution of urethral and urachal urine output.     

Transabdominal chorionic villus biopsy in second and third trimesters of pregnancy to determine fetal karyotype.

Transabdominal chorionic villus biopsy is an established method of obtaining material for analysing fetal chromosomes in the first trimester of pregnancy but has not been widely used for karyotyping in the second and third trimesters, when rapid results are required. The technique was evaluated in two groups of patients, comprising 106 at risk of having a fetus with chromosomal anomalies (105) or X linked disease (one) studied between 13 and 22 weeks (median 15 weeks) of gestation (group 1) and 21 with abnormal fetal findings on ultrasonography studied between 13 and 38 weeks (median 27 weeks) (group 2). Chorionic tissue was collected at the first attempt in 109 patients and at the second attempt in a further 17 independent of the position of the placenta. In one case from group 1 sufficient material for analysis could not be obtained. Seven abnormal karyotypes (six in group 1 and one in group 2) were diagnosed. Karyotyping was unsuccessful in two cases in group 1 (at 17 and 18 weeks'' gestation) and in two in group 2 (at 29 and 38 weeks'' gestation). Follow up of group 1 four weeks after sampling showed no signs of adverse fetal development apart from one unexplained intrauterine fetal death. The findings suggest that chorionic sampling is a safe and valuable additional technique for the late detection of chromosomal defects.     

The role of ACE2, angiotensin-(1–7) and Mas1 receptor axis in glucocorticoid-induced intrauterine growth restriction

Background

Plasma and urine levels of the potent vasodilator Ang-(1–7) are elevated in mid and late pregnancy and are correlated with elevated placental angiogenesis, fetal blood flow, and rapid fetal growth. We hypothesized that Ang-(1–7), its receptor (Mas1) and the enzymes involved in Ang-(1–7) production (ACE2 and Membrane metallo-endopeptidase; MME) are down regulated in response to glucocorticoid administration contributing to IUGR.

Methods

Pregnant female Sprague–Dawley rats were injected with dexamethasone (DEX; 0.4 mg/kg/day) starting from 14 day gestation (dg) till sacrifice at 19 or 21 dg while control groups were injected with saline (n?=?6/group). The gene and protein expression of ACE2, MME, Ang-(1–7) and Mas1 receptor in the placental labyrinth (LZ) and basal zones (BZ) were studied.

Results

DEX administration caused a reduction in LZ weight at 19 and 21 dg (p?<?0.001). IUGR, as shown by decreased fetal weights, was evident in DEX treated rats at 21 dg (p?<?0.01). ACE2 gene expression was elevated in the LZ of control placentas at 21 dg (p?<?0.01) compared to 19 dg and DEX prevented this rise at both gene (p?<?0.01) and protein levels (p?<?0.05). In addition, Ang-(1–7) protein expression in LZ was significantly reduced in DEX treated rats at 21 dg (p?<?0.05). On the other hand, Mas1 and MME were upregulated in LZ at 21 dg in both groups (p?<?0.05 and p?<?0.001, respectively).

Conclusion

The results of this study indicate that a reduced expression of ACE2 and Ang-(1–7) in the placenta by DEX treatment may be responsible for IUGR and consequent disease programming later in life.