Differential influence of the 4F2 heavy chain and the protein related to b(0,+) amino acid transport on substrate affinity of the heteromeric b(0,+) amino acid transporter.

We provide evidence here that b(0,+) amino acid transporter (b(0, +)AT) interacts with 4F2 heavy chain (4F2hc) as well as with the protein related to b(0,+) amino acid transporter (rBAT) to constitute functionally competent b(0,+)-like amino acid transport systems. This evidence has been obtained by co-expression of b(0, +)AT and 4F2hc or b(0,+)AT and rBAT in human retinal pigment epithelial cells and in COS-1 cells. The ability to interact with 4F2hc and rBAT is demonstrable with mouse b(0,+)AT as well as with human b(0,+)AT. Even though both the 4F2hc x b(0,+)AT complex and the rBAT x b(0,+)AT complex exhibit substrate specificity that is characteristic of system b(0,+), these two complexes differ significantly in substrate affinity. The 4F2hc x b(0,+)AT complex has higher substrate affinity than the rBAT x b(0,+)AT complex. In situ hybridization studies demonstrate that the regional distribution pattern of mRNA in the kidney is identical for b(0,+)AT and 4F2hc. The pattern of rBAT mRNA expression is different from that of b(0,+)AT mRNA and 4F2hc mRNA, but there are regions in the kidney where b(0,+)AT mRNA expression overlaps with rBAT mRNA expression as well as with 4F2hc mRNA expression.

Cloning and functional characterization of a Na(+)-independent, broad-specific neutral amino acid transporter from mammalian intestine.

We have isolated a cDNA from a rabbit intestinal cDNA library which, when co-expressed with the heavy chain of the human 4F2 antigen (4F2hc) in mammalian cells, induces system L-like amino acid transport activity. This protein, called LAT2, consists of 535 amino acids and is distinct from LAT1 which also interacts with 4F2hc to induce system L-like amino acid transport activity. LAT2 does not interact with rBAT, a protein with a significant structural similarity to 4F2hc. The 4F2hc/LAT2-mediated transport process differs from the 4F2hc/LAT1-mediated transport in substrate specificity, substrate affinity, tissue distribution, interaction with D-amino acids, and pH-dependence. The 4F2hc/LAT2-associated transport process has a broad specificity towards neutral amino acids with K(t) values in the range of 100-1000 microM, does not interact with D-amino acids to any significant extent, and is stimulated by acidic pH. In contrast, the 4F2hc/LAT1-associated transport process has a narrower specificity towards neutral amino acids, but with comparatively higher affinity (K(t) values in the range of 10-20 microM), interacts with some D-amino acids with high affinity, and is not influenced by pH. LAT2 is expressed primarily in the small intestine and kidney, whereas LAT1 exhibits a much broader tissue distribution.

Human Na(+)-dependent vitamin C transporter 1 (hSVCT1): primary structure, functional characteristics and evidence for a non-functional splice variant.

We report here on the cloning and functional characterization of human Na(+)-dependent vitamin C transporter 1 (SVCT1). The human SVCT1 cDNA, obtained from a Caco2 cell cDNA library, encodes a protein of 598 amino acids with 12 putative transmembrane domains. The SVCT1-specific transcript, 2.4 kb in size, is expressed in kidney, liver, small intestine, thymus and prostate. When expressed heterologously in HRPE cells, SVCT1 mediates the transport of ascorbate, the reduced form of vitamin C, in a Na(+)-dependent manner. The transporter is specific for ascorbate with a K(t) of approximately 75 microM. The relationship between the cDNA-specific uptake rate of ascorbate and Na(+) concentration is sigmoidal with a Na(+):ascorbate stoichiometry of 2:1, indicating that the transport process is electrogenic. In Caco2 cells and in normal human intestine, SVCT1 also exists as a non-functional splice variant with a four amino acid sequence inserted between E-155 and V-156. The splice variant results from the use of a donor site 12 bp downstream of the normal donor site.

Fetal acoustic stimulation in early labor and pathological fetal acidemia: a preliminary report.

OBJECTIVE: To determine if a nonreactive response to fetal acoustic stimulation in early labor can predict a significantly higher risk of umbilical arterial pH <7.10 or <7.00. METHODS: Fetal acoustic stimulation was applied to the fetuses of term parturients (gestational age > or =37 weeks) with cervical dilation of < or =5 cm. The responses to stimulation were correlated with cesarean delivery for fetal distress and umbilical arterial pH. Student's t-test, Chi-square, and Fisher exact test were used; P < 0.05 was considered significant. Relative risks (RR) and 95% confidence intervals (CI) were calculated. RESULTS: The study population contained 271 subjects, of which 90% (244) had a reactive response following acoustic stimulation and 10% (27) a nonreactive response. The maternal demographics, time interval from stimulation to delivery (8.3 +/- 8.7 vs. 8.3 +/- 8.4 h; P = 1.00) were similar in the two groups. Compared to those with a reactive response, patients with a nonreactive response had a significantly greater risk for: 1) cesarean delivery for fetal distress (2.0% vs. 11.1%; P = 0.03, RR 4.1, 95% Cl 1.5, 60.5), 2) umbilical arterial pH <7.10 (2.0% vs. 14.8%; P = 0.007, RR 5.0, 95% CI 2.2, 11.6), and 3) umbilical arterial pH <7.00 (0.8% vs. 7%; P = 0.05, RR 5.0, 95% CI 1.8, 15.2). CONCLUSION: A nonreactive response to fetal acoustic stimulation in early labor is associated with a significantly increased risk for cesarean delivery for fetal distress and neonatal acidosis. This finding extends the potential value of acoustic stimulation as an intrapartum admission screening test.

Molecular and functional characterization of the intestinal Na+-dependent multivitamin transporter.

We have cloned a Na+-dependent multivitamin transporter from rabbit intestine (riSMVT). The cDNA codes for a protein of 636 amino acids with 12 putative transmembrane domains. When expressed in mammalian cells, the cDNA induces Na+-dependent uptake of the vitamins pantothenate and biotin. Lipoate is also a substrate for the cDNA-induced uptake process. The affinity constant for the cDNA-specific transport of pantothenate and biotin is approximately 2 and approximately 8 microM, respectively. The Na+:vitamin stoichiometry is greater than 1, indicating that the transport process is electrogenic. The SMVT-specific transcripts of 3.2 kbp are equally distributed throughout the small intestine. We have also cloned SMVT from the human intestinal cell line Caco-2. The Caco-2 SMVT cDNA codes for a protein of 635 amino acids which is homologous to riSMVT and is identical to the SMVT expressed in the human choriocarcinoma cell line JAR. Caco-2 SMVT also catalyzes Na+-dependent uptake of pantothenate, biotin, and lipoate. In oocytes expressing Caco-2 SMVT, all three vitamins evoke inward currents, confirming the electrogenicity of the transport process.

Human placental Na+-dependent multivitamin transporter. Cloning, functional expression, gene structure, and chromosomal localization.

We have cloned the human Na+-dependent multivitamin transporter (SMVT), which transports the water-soluble vitamins pantothenate, biotin, and lipoate, from a placental choriocarcinoma cell line (JAR). The cDNA codes for a protein of 635 amino acids with 12 transmembrane domains and 4 putative sites for N-linked glycosylation. The human SMVT exhibits a high degree of homology (84% identity and 89% similarity) to the rat counterpart. When expressed in HRPE cells, the cDNA-induced transport process is obligatorily dependent on Na+ and accepts pantothenate, biotin, and lipoate as substrates. The relationship between the cDNA-specific uptake rate of pantothenate or biotin and Na+ concentration is sigmoidal with a Na+:vitamin stoichiometry of 2:1. The human SMVT, when expressed in Xenopus laevis oocytes, induces inward currents in the presence of pantothenate, biotin, and lipoate in a Na+-, concentration-, and potential-dependent manner. We also report here on the structural organization and chromosomal localization of the human SMVT gene. The SMVT gene is approximately 14 kilobase pairs in length and consists of 17 exons. The SMVT gene is located on chromosome 2p23 as evidenced by somatic cell hybrid analysis and fluorescence in situ hybridization.

Cloning and functional expression of a cDNA encoding a mammalian sodium-dependent vitamin transporter mediating the uptake of pantothenate, biotin, and lipoate.

Previous studies have shown that a Na+-dependent transport system is responsible for the transplacental transfer of the vitamins pantothenate and biotin and the essential metabolite lipoate. We now report the isolation of a rat placental cDNA encoding a transport protein responsible for this function. The cloned cDNA, when expressed in HeLa cells, induces Na+-dependent pantothenate and biotin transport activities. The transporter is specific for pantothenate, biotin, and lipoate. The Michaelis-Menten constant (Kt) for the transport of pantothenate and biotin in cDNA-transfected cells is 4.9 +/- 1.1 and 15.1 +/- 1.2 microM, respectively. The transport of both vitamins in cDNA-transfected cells is inhibited by lipoate with an inhibition constant (Ki) of approximately 5 microM. The nucleotide sequence of the cDNA (sodium-dependent multivitamin transporter (SMVT)) predicts a protein of 68.6 kDa with 634 amino acids and 12 potential transmembrane domains. Protein data base search indicates significant sequence similarity between SMVT and known members of the Na+-dependent glucose transporter family. Northern blot analysis shows that SMVT transcripts are present in all of the tissues that were tested. The size of the principal transcript is 3.2 kilobases. SMVT represents the first Na+-dependent vitamin transporter to be cloned from a mammalian tissue.

Vibroacoustic stimulation and fetal behavioral state in normal term human pregnancy.

Vibroacoustic stimulation may affect human fetal behavior. Continuous graphic records of simultaneous ultrasonographic observations of fetal activities and electronic fetal heart rate tracings of 30 normal term fetuses were examined visually for the occurrence of behavioral state 30 minutes before and after 3 seconds of vibroacoustic stimulation. After vibroacoustic stimulation, the total time spent in state 1 decreased significantly, that spent in state 4 increased significantly, and times spent in state 2 and indeterminate state (no state established for at least 3 minutes) were unchanged. No fetus exhibited state 3 before or after vibroacoustic stimulation. State 4 occurred in 22 fetuses after vibroacoustic stimulation with a duration of at least 30 minutes in four fetuses, and was noted in all fetuses in pre-vibroacoustic stimulation state 1 and 11 of 16 fetuses in pre-vibroacoustic stimulation state 2. Fetal heart rate accelerations occurred within 10 seconds after vibroacoustic stimulation in 94% of the fetuses studied regardless of their prior behavioral state. The variation in the onset and duration of behavioral state responses in most fetuses after vibroacoustic stimulation may depend on previous behavioral state and could be important for interpretation of antenatal assessment that uses this stimulus.

The effects of vibroacoustic stimulation on baseline heart rate, breathing activity, and body movements of normal term fetuses.

To determine the fetal biophysical effects of vibroacoustic stimulation produced by an electronic artificial larynx we studied 20 normal term pregnancies assigned either to control (no stimulus) or experimental (stimulus) groups. Each fetus was observed for 3 hours; either no stimulus or a 3-second stimulus was delivered after the first hour. Fetal heart rate baseline and variation, breathing movement incidence, rate, and variation, and body movement incidence data were acquired concurrently and analyzed at 15-minute intervals. Intergroup comparisons showed that, after stimulation, fetal heart rate baseline and variation increased significantly, whereas breathing incidence fell during the first 15 minutes. Within-group analyses showed that poststimulus elevation of fetal heart rate baseline was the only significant time interaction over the 3 hours. Vibroacoustic stimulation appears to be primarily associated with transient alterations in fetal heart rate baseline; concomitant changes in breathing activity probably reflect normal biologic cycles.

The effects of vibratory acoustic stimulation on baseline fetal heart rate in term pregnancy.

Fetal heart rate responses to vibratory acoustic stimulation have been studied in normal and complicated pregnancies. To determine the precise nature of these responses, we studied 50 normal term fetuses with 60 minutes of electronic antepartum fetal heart rate monitoring divided in two 30-minute segments separated by 3 seconds of vibratory acoustic stimulation. All tracings were analyzed by a programmed microcomputer. Comparison of grouped mean 30-minute values before and after vibratory acoustic stimulation showed significant increases in baseline fetal heart rate, fetal heart rate variation, frequency of accelerations exceeding 10 and 15 beats/min, duration of accelerations exceeding 15 beats/min, and frequency of decelerations after vibratory acoustic stimulation. Mean baseline fetal heart rate elevation greater than 10 beats/min occurred within 7.6 +/- 4.4 seconds in 42 of 50 fetuses and lasted for 596 +/- 531 seconds. Reactive tests increased from 35 (70%) to 47 (94%) after vibratory acoustic stimulation. Most healthy term fetuses exhibit fetal heart rate responses after differing in frequency or magnitude from spontaneous fetal heart rate changes. Vibratory acoustic stimulation may be a valid fetal assessment tool but cannot be considered the physiologic equivalent of nonstress testing.