Differential Protein Binding Research Articles

Multiple in vivo footprints are specific to the active allele of the X-linked human hypoxanthine phosphoribosyltransferase gene 5' region: implications for X chromosome inactivation.

Dosage compensation of X-linked genes in male and female mammals is accomplished by random inactivation of one X chromosome in each female somatic cell. As a result, a transcriptionally active allele and a transcriptionally inactive allele of most X-linked genes reside within each female nucleus. To examine the mechanism responsible for maintaining this unique system of differential gene expression, we have analyzed the differential binding of regulatory proteins to the 5' region of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Studies of DNA-protein interactions associated with the transcriptionally active and inactive HPRT alleles were carried out in intact cultured cells by in vivo footprinting by using ligation-mediated polymerase chain reaction and dimethyl sulfate. Analysis of the active allele demonstrates at least six footprinted regions, whereas no footprints were detected on the inactive allele. Of the footprints on the active allele, at least four occur over canonical GC boxes or Sp1 consensus binding sites, one is associated with a potential AP-2 binding site, and another is associated with a DNA sequence not previously reported to interact with a sequence-specific DNA-binding factor. While no footprints were observed for the HPRT gene on the inactive X chromosome, reactivation of the inactive allele with 5-azacytidine treatment restored the in vivo footprint pattern found on the active allele. Results of these experiments, in conjunction with recent studies on the X-linked human PGK-1 gene, bear implications for models of X chromosome inactivation.

Multiple In Vivo Footprints Are Specific to the Active Allele of the X-Linked Human Hypoxanthine Phosphoribosyltransferase Gene 5′ Region: Implications for X Chromosome Inactivation

Dosage compensation of X-linked genes in male and female mammals is accomplished by random inactivation of one X chromosome in each female somatic cell. As a result, a transcriptionally active allele and a transcriptionally inactive allele of most X-linked genes reside within each female nucleus. To examine the mechanism responsible for maintaining this unique system of differential gene expression, we have analyzed the differential binding of regulatory proteins to the 5' region of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Studies of DNA-protein interactions associated with the transcriptionally active and inactive HPRT alleles were carried out in intact cultured cells by in vivo footprinting by using ligation-mediated polymerase chain reaction and dimethyl sulfate. Analysis of the active allele demonstrates at least six footprinted regions, whereas no footprints were detected on the inactive allele. Of the footprints on the active allele, at least four occur over canonical GC boxes or Sp1 consensus binding sites, one is associated with a potential AP-2 binding site, and another is associated with a DNA sequence not previously reported to interact with a sequence-specific DNA-binding factor. While no footprints were observed for the HPRT gene on the inactive X chromosome, reactivation of the inactive allele with 5-azacytidine treatment restored the in vivo footprint pattern found on the active allele. Results of these experiments, in conjunction with recent studies on the X-linked human PGK-1 gene, bear implications for models of X chromosome inactivation.

Differential binding of human nuclear proteins to Alu subfamilies

Several diagnostic differences that distinguish human Alu subfamilies are clustered just downstream from the B box of the RNA polymerase III promoter; we tentatively refer to this diagnostic region as the DB box. Assuming that this region might determine the relative transcriptional activity of Alu subfamilies, we examined the interaction of nuclear proteins with DB box sequences representing different Alu subfamilies. Gel mobility shift assays suggest the existence of two factors which discriminate among the DB boxes of different Alu subfamilies: 1) An abundant, ca. 50 kd, protein binds more stably to a young 'PV' Alu subfamily (PVS) than to the older major subfamily (MS). 2) Methylation of CpG dinucleotides stimulates the binding of a less abundant, ca. 70 kd, protein to the DB boxes of younger Alu subfamilies.

Fetal and Maternal Placental and Nonplacental Clearances of Metoclopramide in Chronically Instrumented Pregnant Sheep

The placental and nonplacental clearances of metoclopramide were studied in nine chronically instrumented, near-term pregnant sheep using a two-compartment open model. Metoclopramide was administered to the ewe and fetus on separate occasions as an initial iv bolus loading dose followed by a constant-rate infusion, with steady-state maternal and fetal plasma concentrations being obtained by 45min. Following the maternal infusions, metoclopramide reached average steady-state concentrations of 50.0 ± 20.2 ng/mL in the ewe and 27.1 ± 8.6 ng/mL in the fetus, with a mean fetal-to-maternal concentration ratio of 0.57 ± 0.14. The ability of the fetus to eliminate metoclopramide by nonplacental routes appears to be responsible for this ratio being less than unity, rather than differential protein binding and ion-trapping effects. Mean steady-state concentrations were 13.8 ± 4.5 and 253.7 ± 92.1 ng/mL in the ewe and fetus, respectively, after fetal drug administration. Metoclopramide was bound significantly less to fetal (39.5 ± 8.9%) than to maternal (49.5 ± 7.9%) plasma proteins, with values similar to that reported for humans (≈40%). Clearance of metoclopramide across the placenta from the fetus to the ewe (6.2 ± 2.4 L/h/kg) was significantly greater than that in the reverse direction (4.3 ± 1.3 L/h/kg) and accounted for ≈80% of total fetal drug elimination. This may be explained by the higher percentage of fetal cardiac output to the placenta and the flow-limited transfer of this compound. Nonplacental routes (e.g., renal, hepatic) were responsible for only ≈20% of total fetal drug elimination (7.9 ± 2.9 L/h/kg), but, they comprised the major source (≈95%) of total maternal drug clearance (2.9 ± 0.4 L/h/kg). Neither maternal nor fetal drug administration resulted in any consistent or significant cardiorespiratory or behavioral effects in the fetal lamb, even though very high concentrations were obtained in fetal plasma following fetal dosing.

Differential Binding of Hemolymph Proteins from Schistosome-Resistant and -Susceptible Biomphalaria glabrata to Schistosoma mansoni Sporocysts

Humoral factors have been associated with resistance of Biomphalaria glabrata to infection by Schistosoma mansoni. The goal of this study was to determine which serum (cell-free hemolymph) proteins bind to the surface of S. mansoni sporocysts. For this, 125I-labeled serum from schistosome-resistant (10-R2) and -susceptible (M-line) B. glabrata was incubated with sporocysts, washed, and then subjected to SDS-PAGE and autoradiography. Other samples examined included radiolabeled 10-R2 and M-line serum, sporocysts incubated with unlabeled serum followed by incubation with radiolabeled serum, and radiolabeled sporocysts. Results indicated that many polypeptides in the serum from both strains of B. glabrata were radiolabeled. Dominating both profiles were bands in the 90-210-kDa range. However, some differences between the serum of the 2 snail strains were observed with M-line serum having several radiolabeled polypeptides in the 31-40- and 66-85-kDa range that were absent in serum from 10-R2 B. glabrata. When sporocysts were incubated with radiolabeled serum, 3 polypeptides (116, 180, 210 kDa) from both snail strains bound to the surface of the parasite. Further, a 55-kDa polypeptide bound to sporocysts incubated with 10-R2 serum but did not bind to those parasites incubated with M-line serum. Preincubation of sporocysts with unlabeled serum prior to incubation with radiolabeled serum significantly inhibited the uptake of radiolabeled proteins. This differential binding of serum polypeptides from different strains of B. glabrata may be important in determining resistance or susceptibility of the snail to larval schistosome infection.

Differential DNA binding of nuclear proteins to a long terminal repeat region of the MCF13 and Akv murine leukemia viruses

Long terminal repeat (LTR) sequences of murine leukemia viruses (MLVs) have been demonstrated to be mainly responsible for the pathogenic differences in these retroviruses. A region of the LTR which is downstream of the enhancer elements has been shown to contribute both to enhancer activity as well as to disease specificity of MLVs. We have identified protein-DNA complexes generated by this region of a lymphomagenic MLV (MCF13) and one which is nonpathogenic (Akv). One protein-DNA complex we have observed for this region is unique to MCF13 DNA sequences. Detection of protein involved in this unique MCF13 complex in different cell lines revealed that it was ubiquitous.

Placental transfer of thiamine in the human subject: In vitro perfusion studies and maternal-cord plasma concentrations

The transfer of thiamine across human placenta has been studied in an in vitro perfusion system. With the maternal and fetal perfusates not recirculated, the transfer index (ratio of clearance of thiamine to that of l-glucose) toward the fetus was 2.09 ± 0.36, suggesting a mediated transport system. In the reverse direction, the transfer rate approximates that expected from simple diffusion (transfer index 0.78 ± 0.25). Stepwise increases in thiamine concentration in the maternal perfusate were associated with parallel increases in transfer rates up to 500 nmol/L. Above that concentration, the rate of increase declined and the transfer index fell. When the maternal perfusate was recirculated and the fetal circulation was kept open, the thiamine concentration rapidly decreased, reaching a plateau in 30 to 40 minutes at a level about 0.1 to 0.2 of the fetal perfusate. In contrast, the concentration within the placenta greatly exceeded those in the perfusates. The observations indicate an effective transport system directed toward the fetus, saturable at low concentrations and capable of establishing a transplacental gradient. Measurement of thiamine concentrations in 20 paired samples of plasma obtained at term demonstrated a mean (± SD) cord:maternal ratio of 2.5 ± 1.4. Differential protein binding was excluded as a cause of the gradient. The in vivo gradient is probably attributable to placental transport.

Differential protein binding in lymphocytes to a sequence in the enhancer of the mouse retrovirus SL3-3.

An electrophoretic mobility shift assay was used to characterize interactions of nuclear proteins with a DNA segment in the enhancer element of the leukemogenic murine retrovirus SL3-3. Mutation of a DNA sequence of the 5'-TGTGG-3' type decreased transcription in vivo specifically in T-lymphocyte cell lines. Extracts of nuclei from different T-lymphocyte cell lines or cells from lymphoid organs resulted in much higher amounts of complexes in vitro with this DNA sequence than did extracts from other cell lines or organs tested. Differences were also found in the sets of complexes obtained with extracts from the different types of cells. The DNA sequence specificities of the different SL3-3 enhancer factor 1 (SEF1) protein complexes were found to be distinct from those of several other previously identified DNA motifs of the TGTGG type because of differences in several nucleotides critical for binding and because these other DNA motifs could not compete with the identified DNA sequence for binding of SEF1. Limited treatment with several different proteases cleaved the SEF1 proteins such that their DNA-binding domain(s) remained and created complexes with decreased and nondistinguishable electrophoretic mobility shifts and with new properties. These results indicate that the SEF1 proteins have a structure with a flexible and relatively vulnerable hinge region linking a DNA-binding domain(s) to a more variable domain(s) with other functions. We suggest that the binding of SEF1 is an essential factor for the T-cell tropism of SL3-3 and the ability of this virus to cause T-cell lymphomas.

Differential protein binding in lymphocytes to a sequence in the enhancer of the mouse retrovirus SL3-3.

An electrophoretic mobility shift assay was used to characterize interactions of nuclear proteins with a DNA segment in the enhancer element of the leukemogenic murine retrovirus SL3-3. Mutation of a DNA sequence of the 5'-TGTGG-3' type decreased transcription in vivo specifically in T-lymphocyte cell lines. Extracts of nuclei from different T-lymphocyte cell lines or cells from lymphoid organs resulted in much higher amounts of complexes in vitro with this DNA sequence than did extracts from other cell lines or organs tested. Differences were also found in the sets of complexes obtained with extracts from the different types of cells. The DNA sequence specificities of the different SL3-3 enhancer factor 1 (SEF1) protein complexes were found to be distinct from those of several other previously identified DNA motifs of the TGTGG type because of differences in several nucleotides critical for binding and because these other DNA motifs could not compete with the identified DNA sequence for binding of SEF1. Limited treatment with several different proteases cleaved the SEF1 proteins such that their DNA-binding domain(s) remained and created complexes with decreased and nondistinguishable electrophoretic mobility shifts and with new properties. These results indicate that the SEF1 proteins have a structure with a flexible and relatively vulnerable hinge region linking a DNA-binding domain(s) to a more variable domain(s) with other functions. We suggest that the binding of SEF1 is an essential factor for the T-cell tropism of SL3-3 and the ability of this virus to cause T-cell lymphomas.

Differential nuclear protein binding to 5-azacytosine-containing DNA as a potential mechanism for 5-aza-2'-deoxycytidine resistance.

A clonal cell line (56-42) that was stably and exclusively resistant to the toxic effects of the antileukemic agent 5-aza-2'-deoxycytidine (5-aza-CdR) was derived from C3H 10T1/2 C18 cells after multiple treatments with 5-aza-CdR. The 50% lethal dose of 5-aza-CdR for these cells was 1.3 microM, which was 15-fold greater than that for the parental cells. Cell line 56-42 was slightly cross-resistant to the ribo-analog 5-azacytidine, but was sensitive to the nucleoside analog 1-beta-D-arabinofuranosylcytosine and to colcemid. Both parental and resistant cell lines incorporated equimolar amounts of 5-aza-CdR into DNA. Resistance was therefore not due to decreased activation, increased detoxification, or reduced incorporation of the drug. The overall level of cytosine methylation in the resistant clone was 80% lower than the level in the sensitive cells. Therefore, the potential number of hemimethylated sites created by the incorporation of equivalent amounts of 5-aza-CdR into the DNA of the two cell types was much greater in the sensitive cells. Furthermore, 5-azacytosine-substituted DNA from the sensitive cells bound 100% more nuclear protein in the form of highly stable complexes. The incorporation of 5-aza-CdR opposite methylated cytosine residues in DNA of the sensitive cells thus resulted in increased nuclear protein binding at hemimethylated sites. This relative increase in tight-binding protein complexes was shown to occur in living cells and may well disrupt replication and transcription and instigate cell death. The differential binding of proteins to hypomethylated, azacytosine-containing DNA may thus mediate a novel mechanism of drug resistance.

DiFferential Nuclear Protein Binding to 5-Azacytosine-Containing DNA as a Potential Mechanism for 5-Aza-2′-Deoxycytidine Resistance

A clonal cell line (56-42) that was stably and exclusively resistant to the toxic effects of the antileukemic agent 5-aza-2'-deoxycytidine (5-aza-CdR) was derived from C3H 10T1/2 C18 cells after multiple treatments with 5-aza-CdR. The 50% lethal dose of 5-aza-CdR for these cells was 1.3 microM, which was 15-fold greater than that for the parental cells. Cell line 56-42 was slightly cross-resistant to the ribo-analog 5-azacytidine, but was sensitive to the nucleoside analog 1-beta-D-arabinofuranosylcytosine and to colcemid. Both parental and resistant cell lines incorporated equimolar amounts of 5-aza-CdR into DNA. Resistance was therefore not due to decreased activation, increased detoxification, or reduced incorporation of the drug. The overall level of cytosine methylation in the resistant clone was 80% lower than the level in the sensitive cells. Therefore, the potential number of hemimethylated sites created by the incorporation of equivalent amounts of 5-aza-CdR into the DNA of the two cell types was much greater in the sensitive cells. Furthermore, 5-azacytosine-substituted DNA from the sensitive cells bound 100% more nuclear protein in the form of highly stable complexes. The incorporation of 5-aza-CdR opposite methylated cytosine residues in DNA of the sensitive cells thus resulted in increased nuclear protein binding at hemimethylated sites. This relative increase in tight-binding protein complexes was shown to occur in living cells and may well disrupt replication and transcription and instigate cell death. The differential binding of proteins to hypomethylated, azacytosine-containing DNA may thus mediate a novel mechanism of drug resistance.

Differential serum protein binding of benzidine- and benzidine-congener based dyes and their derivatives.

Environmental dyes and their derivatives, some of which are genotoxic, must be transported within the body to the tissues which they affect. One mechanism for this can be observed directly by crossed immunoelectrophoresis (X-IEP). Binding of these chemicals to certain serum proteins changes electrophoretic and immunoprecipitation morphology in X-IEP patterns. This is demonstrated here for four azo dyes derived from benzidine, 3,3'-dimethylbenzidine, and 3,3'-dimethoxybenzidine, and their parent aromatic amines. Direct Red 2 (a 3,3'-dimethylbenzidine-based dye), Direct Blue 15 (a 3,3'-dimethoxybenzidine-based dye), Direct Black 38 (a benzidine-based dye), and Evans Blue (a 3,3'-dimethylbenzidine-based dye) all bound to albumin, alpha 1-lipoprotein, beta-lipoprotein, and hemopexin. Direct Red 2 only slightly affected the mobilities of these proteins. Direct Blue 15 bound also to prealbumin and alpha 1-antichymotrypsin, and degraded C3 globulin. Direct Black 38 and Evans Blue bound to numerous additional proteins. Evans Blue bound variably to proteins of sera from different individuals, suggesting that there are individual differences in serum protein binding capabilities for these chemicals. Of the three derivatives of the benzidine dyes, only 3,3'-dimethylbenzidine caused changes in X-IEP patterns, indicating its binding to the serum proteins. This chemical differentially affected sub-populations of alpha 1-lipoprotein, either by altering its electrophoretic mobility or inhibiting its recognition by antibodies. Autoradiographic analyses demonstrated the binding of benzidine and 3,3'-dimethylbenzidine to both alpha 1- and beta-lipoproteins.

THE EFFECT OF IMMATURITY ON INTESTINAL HANDLING OF COW'S MILK PROTEINS

To investigate postnatal maturational changes of gut barrier function, we used two small intestinal in vitro models. Microvillus membrane (MVM) preparations were used for studying membrane surface binding and the everted gut sac technique was used for studying mucous coat binding and uptake of 125I-labelled bovine serum albumin (125I-BSA) and betalactoglobulin (125I-BLG) in newborn, preweanling and adult rats. Surface binding of BSA and BLG to MVM was greater from newborn and preweanling rats, compared to adults (BSA, newborns: 2.74±0.53%; adults 1.08±0.17%, p<0.001). Binding however was weak and nonspecific. Contrary to surface binding, mucous coat binding of BSA and BLG to preweanling gut sacs was less than to adult ones, as was uptake (BSA binding: 0.94±0.30 vs 3.06±0.74 μg 125I-protein equivalents /mg mucosal protein, p<0.001; BSA uptake: 0.35±0.17 vs 0.71±0.20, p<0.01). Gut sac binding and uptake were closely correlated in adult and preweanling animals (r=0.76, p<0.001 for BSA; r=0.85, p<0.001 for BLG in preweanlings). These studies suggest that immature animals are binding more cow's milk proteins to their MVM, but that less protein is bound to the mucous coat and taken up by immature gut sacs. Differential handling, mucous coat and MVM binding of food proteins in immature animals may be controlled by mucus layer factors which act to protect the underlying MVM.

Differential binding of nonhistone chromosomal proteins to the putative mouse origin of replication

Ehrlich ascites tumour (EAT) cells were treated with trioxsalen and ultraviolet light to crosslink DNA in vivo. After the treatment initiation of DNA replication can still occur but elongation is blocked by the crosslinks and this leads to the formation of short DNA fragments containing the origin of replication that can be isolated in double-stranded form after S 1 nuclease cutting of the crosslinked DNA (Russev, G. and Vassilev, L. (1982) J. Mol. Biol. 161, 77–87). To assess the affinity of these DNA fragments toward different chromosomal proteins, chromatin was fractionated by SDS-polyacrylamide gel electrophoresis, proteins were transferred to nitrocellulose filters and allowed to interact with in vivo labelled [ 32P]DNA. The autoradiography of the filters showed that the DNA fraction synthesized between crosslinks and containing the putative mouse origin of replication bound preferentially to several nonhistone proteins, the most strongly binding ones having molecular weights of 64, 68, 72 and 150 kDa.

Disposition of maternally-administered methadone and its effects on hepatic drugmetabolizing functions in perinatal guinea pigs

Racemic methadone HC1 (25 mg/kg given every 12 hr for 2 consecutive days) was administered orally to pregnant (60–65 days of gestation) or nursing (0–8 days post-partum) albino guinea pigs. The dams, fetuses and pups were killed 12 hr after the last dose for the anaysis of maternal and perinatal plasma, brain, hepatic and renal methadone residues by GLC and the in vitro measurement of maternal and perinatal microsomal mono-oxygenase [ p-nitroanisole O-demethylase (OD) and aniline hydroxylase (AH)] and glucuronosyltransferase (GT) activities. Methadone was acquired by the perinatal guinea pig both transplacentally and via the milk, more agent being obtained by the former route. Fetal plasma levels of methadone were consistently lower than those detected in the mother due, largely, to differential plasma protein binding. The perinatal pattern of tissue distribution of methadone was markedly different from that observed in the dam, exceedingly high levels being found in fetal brain and kidney. Maternally-administered methadone did not affect the ontogenesis of perinatal OD, AH and GT but it did reduce significantly the levels of hepatic GT activity in the pups and in the dams.

Placental Transfer of Ranitidine During Steady-State Infusions of Maternal and Fetal Sheep

The placental transfer of ranitidine was studied at pharmacokinetic steady state in anesthetized, full-term, pregnant sheep. Ranitidine was administered to the ewe in three preparations and to the fetus in three other sheep. In all experiments, dose size was based on the combined maternal-fetal weight. Steady-state plasma levels were reached within 4hr by using an initial intravenous bolus dose followed by continuous infusion. Following maternal dosage, the mean maternal (jugular vein) steady-state concentration (CMss) at 4hr was 842 ± 66 ng/ml (SEM) the mean fetal (carotid artery) steady-state concentration (CFss ) was 26.5 ± 4.2 ng/ml, and the mean fetal umbilical venous steady-state concentration was 28.9 ± 3.5 ng/ml. Both the fetal and umbilical plasma concentrations were significantly less than the maternal plasma concentration (p <0.01). With fetal dosage, mean CMss was 414 ± 42 ng/ml at 4hr and was significantly less than the mean CFss value at the same time, which was 6890 ± 360 ng/ml (p <0.005). Ranitidine was not bound extensively to plasma proteins in the ewe or the fetus (range 12-55% bound). The reversal of the CMss∕CFss gradient with the change from maternal to fetal administration and the low binding of the drug shows that the gradient following maternal dosage cannot be explained by ion-trapping or differential plasma protein binding. As active placental transport is considered unlikely, the low fetal plasma concentrations are probably due to the presence of significant fetal elimination of ranitidine. Furthermore, the substantial gradient between maternal and umbilical venous plasma concentrations suggests that placental elimination of ranitidine should also be considered.

Distribution of Dexamethasone between Mother and Fetus after Maternal Administration

FUNKHOUSER, J. D.; PEEVY, K. J.; MOCKRIDGE, P. B. and; HUGHES, E. R. Author Information

Interactions of contractile proteins with free and immobilized Cibacron Blue F3GA

Differential binding of contractile proteins from skeletal muscle to Cibacron Blue F3GA-Sepharose affinity columns provides the basis for a new purification technique. Myosin subfragments bind at low ionic strength and are eluted by high salt (e.g., 1.5 m NaCl). Myosin light chain 2 also binds at low ionic strength, whereas light chain 1 is only partially retarded and light chain 3 does not bind. Myosin's marginal solubility in the low-salt buffers required for binding renders it unsuitable for Blue Sepharose chromatography. Neither G-actin nor F-actin bind. Crude preparations of myosin subfragment-1 or light chains undergo significant purification upon Blue Sepharose chromatography. Nee free chromophore inhibits the ATPase activities of myosin and actomyosin at micromolar dye concentrations, whereas the binding of subfragment-1 to actin (in myofibrils) and the tension of glycerinated fibers are inhibited at millimolar dye concentrations. The dye binds at multiple sites on myosin, and inhibits its actomyosin ATPase both competitively and uncompetitively.

Antischizophrenic drugs: Differential plasma protein binding and therapeutic activity

The percentage of free neuroleptic drug unbound to plasma protein is much higher for the respective metabolites of chlorpromazine and thioridazine, 7-hydroxychlorpromazine and mesoridazine, than for the parent drugs. The therapeutic activities of chlorpromazine and thioridazine may be mediated to a major extent by 7-hydroxychlorpromazine and mesoridazine respectively. Measuring free levels of the active metabolites of neuroleptics as well as the parent drugs may facilitate regulation of neuroleptic doses to secure optimal therapeutic responses.

Distribution of dexamethasone between mother and fetus after maternal administration.

Summary: The adrenal glucocorticoid hormones are believed to have an important role in the definitive biochemical differentiation of a number of fetal organs. This paper presents data for dexamethasone transfer and distribution between mother and fetus using 20-21 days pregnant rats. This synthetic glucocorticoid is rapidly transferred to the fetus and reaches a plateau by 90 min after administration to the mother. Pregnant rats receiving either a high dose (20 mg) or a low dose (2.2 µg) had almost identical concentration distribution ratios with a mean of 0.311 (± 0.013) and 0.332 (± 0.019) between fetal and maternal plasmas, respectively. In vitro binding measured by equilibrium dialysis showed that maternal plasma bound only 4-5% more than the fetal plasma. Essentially no radioactivity remained bound to protein after Sephadex G-50 chromatography, indicating no high affinity sites for dexamethasone in either maternal or fetal plasma. Acetylation of ethyl acetate extracts of fetal and maternal plasma followed by paper chromatograph revealed two identical peaks for both the dexamethasone standard and the plasma radioactivity. This study demonstrates a rapid transfer of administered dexamethasone from the mother to the fetus with a concentration difference between the two plasma pools. The difference in concentration does not appear to be the result of differential protein binding or metabolism. Speculation: The rat placenta would appear to maintain an appreciable concentration difference between maternal and fetal plasma for the glucocorticoid hormones. The nature of the process which accounts for this phenomenon needs further investigation.