Mechanism of foetal wastage following immunoneutralization of riboflavin carrier protein in the pregnant rat: disturbances in flavin coenzyme levels

Immunoneutralization of maternal RCP results in a greater than 90% decrease in the content and the incorporation of [2-14C]riboflavin into embryonic FAD as well as a percentage redistribution of both embryonic FMN and riboflavin. This is unaccompanied by any discernible changes in flavin distribution pattern in the maternal liver. Embryonic alpha-glycerophosphate dehydrogenase and NADPH-cytochrome c reductase register significant decreases in activities in the RCP antiserum-treated rats. These alterations readily explain the arrest of foetal growth culminating in pregnancy termination in the antiserum-treated animals.     

Isolation of riboflavin carrier proteins from pregnant human and umbilical cord serum: Similarities with chicken egg riboflavin carrier protein

A riboflavin carrier protein has been purified from human pregnancy and umbilical cord sera by affinity chromatography and fast protein liquid chromatography. This protein has a similar molecular weight to the chicken egg riboflavin carrier protein and shares other physicochemical properties, such as pI and riboflavin binding characteristics, with the avian counterpart. A high degree of immunological cross-reactivity is observed between the human and avian riboflavin binding proteins indicating the extensive conservation of this protein throughout evolution.     

Effect of maternal ethanol consumption on foetal and neonatal rat hepatic protein synthesis.

Effects of maternal ethanol consumption were investigated on the rates of protein synthehsis by livers of foetal and neonatal rats both in vivo and in vitro, and on the activities of enzymes involved in protein synthesis and degradation. The rates of general protein synthesis by ribosomes in vitro studied by measuring the incorporation of [14C]leucine into ribosomal protein showed that maternal ethanol consumption resulted in an inhibition of the rates of protein synthesis by both foetal and neonatal livers from the ethanol-fed group. The rates of incorporation of intravenously injected [14C]leucine into hepatic proteins were also significantly lower in the foetal, neonatal and adult livers from the ethanol-fed group. Incubation of adult-rat liver slices with ethanol resulted in an inhibition of the incorporation of [14C]leucine into hepatic proteins; however, this effect was not observed in the foetal liver slices. This effect of externally added ethanol was at least partially prevented by the addition of pyrazole to the adult liver slices. Pyrazole addition to foetal liver slices was without significant effect on the rates of protein synthesis. Cross-mixing experiments showed that the capacity of both hepatic ribosomes and pH5 enzyme fractions to synthesize proteins was decreased in the foetal liver from the ethanol-fed group. Maternal ethanol consumption resulted in a decrease in hepatic total RNA content, RNA/DNA ratio and ribosomal protein content in the foetal liver. Foetal hepatic DNA content was not significantly affected. Ethanol consumption resulted in a significant decrease in proteolytic activity and the activity of tryptophan oxygenase in the foetal, neonatal and adult livers. It is possible that the mechanisms of inhibition of protein synthesis observed here in the foetal liver after maternal ethanol consumption may be responsible for at least some of the changes observed in 'foetal alcohol syndrome'.     

Enzymic imbalance in serine metabolism in rat hepatomas.

The renaturation of the tetrameric enzyme phosphoglycerate mutase from baker's yeast after denaturation in guanidinium chloride was studied. Three proteinases (trypsin, chymotrypsin and thermolysin) cause extensive loss of activity of samples taken during the early stages of refolding. As judged by SDS/polyacrylamide-gel electrophoresis, the proteinases cause substantial degradation of the polypeptide chain with no evidence for large quantities of fragments of Mr greater than 6500. These data suggest that the early intermediates in the refolding, especially the folded monomer, possess a number of sites that are susceptible to proteolysis.     

Gene and protein expression profiles in the foetal liver of the pregnant rat fed a low protein diet

Foetal growth is particularly sensitive to the protein content of the mother’s diet. Microarray data from the foetal liver of pregnant rats fed normal (HP) or reduced protein diets (LP) were compared by gene set enrichment analysis. Soluble proteins from a second portion of the liver were analysed by two-dimensional gel electrophoresis. Genes associated with progesterone, insulin-like growth factor-1 and vascular endothelial growth factor were upregulated in HP compared to LP, in addition to genes associated with cell differentiation and signalling from the extracellular matrix. In contrast, cytokine signalling was downregulated. Proteomics showed that proteins associated with amino acid metabolism, mitochondrial function and cell motility were differentially abundant in the HP compared to the LP groups. These growth factor and extracellular matrix signalling pathways linked to cell motility may be important mediators of the changes in liver structure that occur in utero and persist into adult life.     

Antigenic determinants on chicken riboflavin carrier protein. A study with monoclonal antibodies

Monoclonal antibodies raised against chicken egg white riboflavin carrier protein were classified into seven categories each recognizing a distinct epitope. Of these, six were directed against conformation dependent epitopes and one to a sequential epitope. The roles of lysine residues and the post-translationally attached phosphate and oligosaccharide moieties in the antigenicity of riboflavin carrier protein recognized by the monoclonal antibodies were investigated. The binding region of three monoclonal antibodies could be located within the 87–219 amino acid sequence of the protein and one antibody among these recognized a sequence of 182–204 amino acid residues. All the monoclonal antibodies were able to recognize riboflavin carrier proteins present in the sera of pregnant rats, cows and humans indicating that the epitopes to which they are directed are conserved through evolution from chicken to the human.     

Folded conformations of antigenic peptides from riboflavin carrier protein in aqueous hexafluoroacetone.

Riboflavin carrier protein (RCP) plays an important role in transporting vitamin B2 across placental membranes, a process critical for maintenance of pregnancy. Association of the vitamin with the carrier protein ensures optimal bioavailability, facilitating transport. The conformations of three antigenic peptide fragments encompassing residues 4-23 (N21), 170-186 (R18), and 200-219 (Y21) from RCP, which have earlier been studied as potential leads toward a synthetic peptide-based contraceptive vaccine, have been investigated using CD and NMR spectroscopy in aqueous solution and in the presence of the structure-stabilizing cosolvent hexafluoroacetone trihydrate (HFA). In aqueous solution at pH 3.0, all three peptides are largely unstructured, with limited helical population for the peptides R18 and Y21. The percentage of helicity estimated from CD experiments is 10% for both the peptides. A dramatic structural transition from an unstructured state to a helical state is achieved with addition of HFA, as evidenced by intensification of CD bands at 222 nm and 208 nm for Y21 and R18. The structural transition is completed at 50% HFA (v/v) with 40% and 35% helicity for R18 and Y21, respectively. No structural change is evident for the peptide N21, even in the presence of HFA. NMR analysis of the three peptides in 50% HFA confirms a helical conformation of R18 and Y21, as is evident from upfield shifts of CalphaH resonances and the presence of many sequential NH/NH NOEs with many medium-range NOEs. The helical conformation is well established at the center of the sequence, with substantial fraying at the termini for both the peptides. An extended conformation is suggested for the N21 peptide from NMR studies. The helical region of both the peptides (R18, Y21) comprises the core epitopic sequence recognized by the respective monoclonal antibodies. These results shed some light on the issue of structure and folding of antigenic peptides.     

Early pregnancy termination in rats immunized with denatured chicken riboflavin carrier protein.

Immunoneutralization of the maternal riboflavin carrier protein in the pregnant rat with antibodies to chicken egg vitamin carrier has earlier been shown to terminate their pregnancies. In order to understand the nature of the epitopic conformations capable of eliciting antibodies bioneutralizing the endogenous riboflavin carrier protein in the pregnant rat, we compared pregnancy progression in the fertile rodents following active immunization with either the native, SDS-denatured, reduced-carboxymethylated or SDS-treated reduced carboxymethylated avian egg white riboflavin carrier protein. The data revealed that despite the total antibody titers being higher in the animals immunized with the native protein, the antibodies elicited against the denatured avian vitamin carrier exhibited relatively better potencies to bioneutralize the endogenous maternal protein as evidenced by higher rates of early fetal resorption.     

Hormonal basis of hysterectomy-induced maternal behavior during pregnancy in the rat.

Hysterectomy during the last half of pregnancy (i.e., Day 10–19) induces a rapid onset of maternal behavior; ovariectomy in addition to hysterectomy, prevents this effect. Estradiol and progesterone were tested for their ability to restore short-latency maternal behavior in hysterectomized-ovariectomized (HO) females operated on the 10th, 13th, 16th and 19th days of pregnancy. A single injection of either 20 μg/kg or 100 μg/kg estradiol benzoate (EB) immediately following HO either alone or followed by 0.5 mg progesterone (P) 44 hr later restored short-latency maternal behavior similar to that observed following hysterectomy only. The lower dose of EB was found to be equally effective at all stages of pregnancy and P was unnecessary to induce maternal behavior. The effectiveness of EB in inducing maternal behavior was discussed in relation to the hormonal changes which follow hysterectomy during pregnancy and to those which are associated with the normal onset of maternal behavior around parturition.     

The metabolism of acetone in the pregnant rat

The administration of a single dose of acetone (100 mg/kg bw) to virgin and 21-day pregnant rats resulted in the appearance of relatively high concentrations of 1,2-propanediol, acetol and methylglyoxal in plasma and liver. In the fetuses no methylglyoxal was detectable. The acetone metabolism curves tend to indicate that the capacity for acetone disposal may be enhanced in the 48 hr-fasted pregnant rat, thus enabling the animal to re-use acetone metabolically, possibly for accelerated gluconeogenesis.     

Termination of pregnancy in mice following antiserum administration to modified chicken riboflavin carrier protein.

Rabbit antibodies to native riboflavin carrier protein (RCP), are to a large extent directed towards conformational epitopes and antibodies to disulphide bond reduced carboxymethylated RCP (RCM-RCP) are towards sequential epitopes. The major cyanogen bromide (CNBr) fragments and tryptic fragments of RCM-RCP interact with both antiserum to RCM-RCP and RCP. Passive immunization of pregnant mice with antibodies to RCM-RCP results in bioneutralization, leading to termination of pregnancy. Recently, a major tryptic fragment of RCM-RCP (24 +/- 2 kd) which could assume conformation at the antibody combining site of native RCP, obtained following mild trypsinization has been identified [Natraj et al. J. Biosci, 15 (1990) 341]. Rabbit antibodies to RCM-RCP treated with trypsin generated antibodies of low titer which interacted with RCM-RCP as well as RCP. The interaction of this antibody with RCP was of high affinity and could be displaced with RCP. The bioneutralizing ability of the antibody was demonstrated by its ability to cause termination of pregnancy in mice.     

The effect of dietary protein on the amino acid supply and threonine metabolism in the pregnant rat

To characterise the effects of dietary protein content on threonine metabolism during pregnancy, rats were fed diets containing 18% or 9% protein and then killed at different stages of gestation. Serum threonine concentrations fell significantly faster in the animals fed the diet containing 9% protein when compared to those fed the diet containing 18% protein. On day 4 of gestation the rate of threonine oxidation was higher in maternal liver homogenates prepared from the animals fed the diet containing 18% protein. The rate of threonine oxidation by liver homogenates fell as gestation proceeded in both diet groups. The activity of threonine dehydrogenase in the maternal liver was unaffected by dietary protein content at all stages of gestation. Serine-threonine dehydratase activity in homogenates of the maternal liver was transiently increased during the early stages of gestation in the animals fed high protein diets but was unchanged in the low protein groups. There was an increase in serine-threonine dehydratase activity in the kidney during the later stages of gestation but this was unaffected by the protein content of the maternal diet. These data show that the changes in free threonine concentrations cannot be accounted for through changes in the oxidation rate and suggest that some other factor influences the unusual metabolism of this amino acid during gestation.