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Biomedical subjects

L K Kelley

Publications and source records attributed to L K Kelley.

At least 19 recordsLinked to original sources
PubMed1995

A method for interactive medical instruction utilizing the World Wide Web.

We describe the implementation of interactive medical teaching programs in radiology and histology which utilize the Internet's World Wide Web (WWW). The WWW standard hypertext interface allows for simple navigation between related documents but does not provide a method for student tracking or question queries. Electronic forms, a recent feature of the WWW, provide the means to present question documents to remote clients and track student performance. A feature of our approach is dynamic creation of HTML documents based upon interaction with database applications. The approach allows multiple simultaneous, yet asynchronous interactions by geographically dispersed students upon the same instructional database and is scalable, providing the capability for multiple image/document servers. The security of the database is assured given that it is not accessible through the Internet.

Computer Communication Networks↗
PubMed1992

Transport and binding in calcium uptake by microvillous membrane of human placenta.

Calcium entry across the microvillous membrane of the human placental syncytiotrophoblast is the first step in the transfer of this important nutrient to the fetus. Calcium uptake by isolated microvillous membranes was time dependent. Equilibrium uptake was very much greater than could be explained by equilibration of the vesicle space with medium, indicating that calcium is bound to internal sites. Addition of the ionophore A23187 greatly increased the rates of influx and efflux, indicating that transport across the plasma membrane is rate limiting in entry. Concentration dependence data for calcium transport at 4 s fit well to a Michaelis-Menten equation having two saturable sites and diffusion. Calcium entry by both transporters was unaffected by calcium channel blockers but was strongly inhibited by the group II metals. The distinct inhibition constant values for strontium inhibition provided additional evidence for two transporters. Calcium binding fit well to a single-site model saturable in the micromolar range. In vivo, the saturable transport processes may mediate calcium entry into the syncytiotrophoblast and binding may regulate concentration within the placental microvilli.

Barium↗
PubMed1990

The calcium-transporting ATPase and the calcium- or magnesium-dependent nucleotide phosphatase activities of human placental trophoblast basal plasma membrane are separate enzyme activities.

The properties of calcium-stimulated ATP hydrolysis often differ from those of ATP-dependent calcium transport. We have characterized two components of calcium-stimulated ATP hydrolysis in human placental basal plasma membrane. In the absence of magnesium, component 1 apparently has saturable sites for free calcium in both the nanomolar and low micromolar range. It was stimulated by either calcium or magnesium, was unselective for nucleotide substrate, and its activity was very much greater than that of ATP-dependent calcium transport. Component 1 was inhibited by GTP, permitting measurement of component 2 with activity and magnesium stimulation comparable to ATP-dependent calcium transport. Component 2 was inhibited partially by an antibody against purified erythrocyte calcium transporter and completely by sulfhydryl reagents, whereas component 1 was unaffected. A phosphorylated intermediate of the calcium transporter co-migrated with the erythrocyte transporter on acidic sodium dodecyl sulfate-polyacrylamide electrophoresis gels. Immunostaining after transfer to nitrocellulose revealed a doublet. The band of lower molecular weight co-migrated with that of the human erythrocyte membrane transporter. The addition of GTP permits separate measurement of ATP hydrolysis by the calcium transporter of the placental basal plasma membrane and may be useful in defining its properties in other cell membranes under a variety of conditions.

Adenosine Triphosphatases↗
PubMed1990

Anionic amino acid transport systems in isolated basal plasma membrane of human placenta.

The placenta absorbs anionic amino acids from the maternal and fetal circulations but does not significantly transfer these amino acids from mother to fetus. Uptake of L-aspartate and L-glutamate by basal (fetal-facing) plasma membrane vesicles from placental syncytiotrophoblast was stimulated by an inward sodium and an outward potassium gradient. Measurable saturable uptake was entirely sodium dependent and electrogenic. Studies of concentration dependence resolved a high-affinity (microM) system that has characteristics of the X-AG system found in other tissues including the placental microvillous plasma membrane. Uptake of 0.2 microM L-glutamate was inhibited by 2 mM L-glutamate, L-aspartate, D-aspartate, L-cysteate, and L-cysteinesulfinic acid and was uninhibited by 2 mM D-glutamate, L-glutamine, L-alanine, L-serine, L-asparagine, and taurine or by 1 mM methylaminoisobutyric acid. The X-AG system in the two membranes of the placental syncytiotrophoblast may mediate the concentrative uptake of anionic amino acids from the maternal and fetal circulations into the placenta.

Amino Acids↗
PubMed1989

Calcium pump epitopes in placental trophoblast basal plasma membranes.

The syncytiotrophoblast represents the primary cellular barrier between maternal and fetal circulations in the placenta. Large amounts of Ca2+ are transported across this barrier by mechanisms that are not clearly understood. To further understand this phenomenon, we examined rat and human placenta by immunohistochemical and protein blotting techniques with a monoclonal antibody raised against the human erythrocyte plasma membrane Ca2+ pump. Immunohistochemistry with this antibody showed specific staining in the human placenta of the basal (fetal facing) surface of the syncytiotrophoblast. In the rat placenta, immunohistochemistry also showed specific staining of the innermost (fetal facing) layer of the trophoblast and the basal surface of the endoderm of the intraplacental yolk sac. In Western blots of placental homogenates and membranes, the monoclonal antibody bound to a 140,000-mol wt band, characteristic of Ca2+ pumps in other tissues. Western blots of isolated basal membranes showed more intense staining than isolated microvillous membranes, confirming the results of the immunohistochemistry. In addition, Ca2+ transport in basal membrane vesicles from human placenta was inhibited by polyclonal antibodies prepared against the erythrocyte Ca2+ pump. We conclude that basal (fetal facing) layers of human and rat placentas contain a high-affinity Ca2+ pump situated to transport Ca2+ from the maternal to the fetal circulation.

Animals↗
PubMed1988

The apical and basal plasma membranes of the human placental syncytiotrophoblast contain different erythrocyte membrane protein isoforms. Evidence for placental forms of band 3 and spectrin.

Using immunochemical techniques, we identified forms of erythrocyte membrane proteins in apical and basal plasma membranes of human placental trophoblast. A wheat germ agglutinin-binding intrinsic protein was present in the microvillous (maternal facing) but not the basal (fetal facing) membrane of the syncytiotrophoblast epithelium. Conversely, erythrocyte-related proteins of the basal membrane included two intrinsic membrane proteins, a 95,000 Mr band 3 isoform and a form of spectrin. These four proteins were all absent from the microvillous membrane. The basal membrane spectrin isoform was also present in basal membrane skeletons. A 70,000 Mr polypeptide which reacted with antibodies to band 3 was present in both microvillous and basal plasma membranes. Therefore, certain isoforms of red cell membrane proteins are polarized between the two surfaces of the human placental syncytiotrophoblast. We propose that the localization of spectrin to the basal membrane is related to the less bundled organization of microfilaments at this membrane compared with that of the microvillous membrane. The band 3 isoforms are candidates for participation in maternofetal anion transport.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1987

Use of GTP to distinguish calcium transporting ATPase activity from other calcium dependent nucleotide phosphatases in human placental basal plasma membrane.

In many cells other than the erythrocyte, the relationship between ATP dependent calcium transport and calcium dependent ATP hydrolysis is complex. The characteristics of ATP hydrolysis often differ from those of calcium transport. Demonstration of a specific transport ATPase is complicated by heterogeneity and high background activity in the presence of magnesium. In basal plasma membrane of human placental syncytiotrophoblast, the addition of 5 mM GTP greatly reduces the background release of 32Pi from 0.1 mM [gamma, 32P]-ATP. The addition of GTP permits measurement of high affinity calcium dependent ATPase under conditions which support calcium uptake. GTP does not affect the velocity of calcium uptake, and in its presence the calcium and magnesium concentration dependence of calcium uptake and calcium dependent ATPase are similar.

Calcium↗
PubMed1987

ATP-dependent calcium transport across basal plasma membranes of human placental trophoblast.

As a first step in understanding the cellular basis of maternal-fetal calcium transfer, we examined the characteristics of calcium uptake by a highly purified preparation of the syncytiotrophoblast basal (fetal facing) plasma membrane. In the presence of nanomolar concentrations of free calcium, basal membranes demonstrated substantial ATP-dependent calcium uptake [K0.5 = 119 nM, maximum velocity (Vmax) = 2 nM X min-1 X mg-1]. This uptake required magnesium, was not significantly affected by Na+ or K+ (50 mM), or sodium azide (10 mM). Intravesicular calcium was rapidly and completely released by the calcium ionophore A23187. Calcium transport was significantly stimulated by the calcium-dependent regulatory protein calmodulin. Placental membrane fractions enriched in endoplasmic reticulum (ER) and mitochondria also demonstrated ATP-dependent calcium uptake. In contrast to basal membrane, mitochondrial calcium uptake was completely inhibited by azide. The rate of calcium uptake by the ER was only 20% of that of basal membranes. We conclude that the placental basal plasma membrane possesses a high-affinity calcium transport system similar to that found in plasma membranes of a variety of cell types. This transporter is situated to permit it to function in vivo in maternal-fetal calcium transfer.

Adenosine Triphosphate↗
PubMed1986

Identification of a novel Mr = 76-kDa form of beta-adrenergic receptors.

The structure of the human beta-adrenergic receptor in purified basal membranes of human placental syncytiotrophoblast was probed using photoaffinity labeling. Basal membranes display a high specific activity of receptors (4-5 pmol/mg protein) and possess both beta 1- and beta 2-adrenergic receptors subtypes. Autoradiography of membranes that were incubated with the beta-adrenergic antagonist [125I]iodoazidobenzylpindolol, photolyzed and then subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, identified four radiolabeled peptides, Mr = 65-kDa, 54-kDa, 43-kDa and a novel higher molecular weight 76-kDa form of the receptor. Photoaffinity labeling of each of these four peptides displayed the pharmacological properties expected for true beta-adrenergic receptors. The 76-kDa photoaffinity labeled receptor peptide observed in human placenta basal membranes has not been reported elsewhere. Competition studies with the beta1-selective ligand CGP-20712A demonstrate that the photoaffinity labeled receptor peptides are composed of both beta 1- and beta 2-adrenergic receptor subtypes.

Adrenergic beta-Antagonists↗
PubMed1983

Isolation and partial characterization of the basal cell membrane of human placental trophoblast.

The function of the syncytiotrophoblast in maternal-fetal exchange is related to the properties of its microvillous (maternal-facing) and basal (fetal-facing) plasma membranes. We have previously reported the properties of the microvillous membrane (Smith, C.H., Nelson, D.M., King, B.F., Donohue, T.M., Ruzycki, S.M. and Kelley, L.K. (1977) Am. J. Obstet. Gynecol. 128, 190-196), and now describe the purification and partial characterization of the basal plasma membrane. Sonication and incubation with EDTA were used to isolate selectively the basal cell membrane. These steps were followed by a more conventional purification by centrifugation. The trophoblast was disrupted and its microvillous membrane and cytoplasmic contents were removed by sonication. The exposed basal cell membrane was selectively released from the underlying basal lamina by sonication in the presence of EDTA and further purified by discontinuous Ficoll gradient centrifugation. The material at the 4-10% Ficoll interface consisted of smooth membrane vesicles with internal microfilaments. It was 45-fold enriched in dihydroalprenolol binding activity and 11-fold enriched in ouabain binding activity. Other enzymatic analyses, including alkaline phosphatase, cytochrome-c oxidase, cytochrome-c reductase and galactosyl transferase indicated low contamination by other organelles. This procedure yields a preparation of relatively high purity which should be suitable for investigation of transport and other functions of the basal surface membrane of trophoblast. In principle, the purification procedures used may be applicable to other transporting epithelia.

Alkaline Phosphatase↗
PubMed1983

Protein kinases of the human placental microvillous membrane. Their potential role in intrasyncytial communication.

Protein phosphorylation has been shown to alter various plasma membrane functions. To investigate the role of phosphorylation in human placental trophoblast, microvillous membrane vesicles were incubated with [gamma-32-P]ATP and the phosphorylation of endogenous and exogenous protein substrates was measured. The microvillous membrane was shown to possess both adenosine 3',5'-cyclic monophosphate (cAMP)-independent and cAMP-dependent kinases. Both endogenous proteins and exogenous proteins were phosphorylated and these processes were enhanced by the presence of Triton or the ionophore alamethicin. The phosphorylation of histone and of endogenous peptides of molecular weights (MW) 147 000, 97 000 and 53 000 was increased by the addition of cAMP. cAMP stimulation required the presence of Triton or alamethicin. The cAMP-dependent kinases are apparently located at the internal (cytoplasmic) surface of the membrane. This location would allow stimulation by cAMP produced by the basal (fetal-facing) plasma membrane. cAMP-stimulated protein phosphorylation may serve as a means of communication between the syncytial plasma membranes facing the fetal and maternal surfaces.

Alamethicin↗
PubMed1982

Placental amino acid uptake. VI. Regulation by intracellular substrate.

Amino acid uptake by human placental tissue is regulated by intracellular amino acids. alpha-Aminoisobutyric acid (AIB) uptake was reduced at intracellular AIB concentrations of 0.8 mM. The magnitude of reduction increased sharply between 1 and 3 mM and reached a maximum of 45% at 5 mM. Suppression was specific to the "A" system. It occurred only when both the amino acid used for preloading and that used as an uptake substrate were active with that system. In the "L" system, facilitation apparently occurs, and in the "ASC" system there is no apparent effect. The system specificity as well as other evidence indicated that suppression is caused by substrate present intracellularly rather than by dilution of extracellular substrate. Suppression was independent of inhibitors of protein synthesis and was not seen in membrane vesicles prepared from preloaded tissue, indicating that intracellular substrate interacts directly with the carrier (transinhibition) rather than altering its synthesis or degradation. The A system transinhibition has the potential to regulate syncytial uptake in vivo and limit variation due to changes in maternal plasma amino acid concentration.

Amino Acids↗
PubMed1980

Placental amino acid uptake. V. Relationship to placental maturation in the rat.

The functional maturation of the placenta during the latter portion of pregnancy is almost certainly essential to fetal growth but its mechanism is largely unknown. To determine the role of changes in intrinsic cellular transport in this process we measured the activity of transport systems for AIB between day 14 and term, a period of known marked increase in in vivo AIB transfer in the rat. In vitro incubation demonstrated that the labyrinthine tissue possessed two transport systems for cellular AIB uptake. Their maximum velocities remained essentially constant from day 16 to term and the intracellular concentration achieved during incubation actually decreased with gestational and the intracellular concentration achieved during incubation actually decreased with gestational age. In vitro tissue preincubation increased cellular uptake of AIB and this response also decreased with maturation. Thus changes in intrinsic transport mechanisms do not at all parallel the very large maturational increase in in utero amino acid transfer. Changes in intrauterine factors such as blood flow, the hormonal millieu, or fetal utilization and the resultant placental-fetal concentration gradients are much more likely to account for the increase in transfer than are alterations in cellular transport mechanisms.

Amino Acids↗
PubMed1978

Placental amino acid uptake. IV. Transport microvillous membrane vesicles.

The recent characterization of a vesicular microvillous plasma membrane preparation from human placenta has made possible study of the characteristics of amino acid transport by the isolated membrane. Variation of medium osmolality demonstrated that alpha-aminoisobutyric acid (AIB) is taken up virtually entirely into a vesicular space. Vesicular volume was determined from equilibrium uptake of three dissimilar nonamino acid substrates, all of which gave a common value. Temperature dependence, saturability, specificity, and similarity of kinetic constants all indicated that AIB uptake by the vesicles resembles that by whole villous tissue. AIB uptake was stimulated specifically by an inward sodium gradient and unaffected by an outward potassium gradient. The persistence of stimulation up to 2 h after the gradient was established could be correlated with the slow equilibration of sodium across the membrane. AIB exodus could be studied with minimal problems of substrate recapture by using preloaded membrane vesicles bound tightly to cellulose ester filters to facilitate transfer to fresh medium. The exodus process was saturable with a Km considerably greater than that for entry.

Alkaline Phosphatase↗
PubMed1977

Characterization of a microvillous membrane preparation from human placental syncytiotrophoblast: a morphologic, biochemical, and physiologic, study.

The syncytiotrophoblast microvillous membrane must play a vital role in many essential functions of the placenta. In order to better understand the functional characteristics of this membrane, we have investigated an isolated membrane preparation by a variety of techniques. Electron microscopic observations showed membranous structures similar to microvilli of intact placental villi in size, shape, and microfilamentous content. Similarities in colloidal iron staining and transferrin localization were also shown. The preparation was enriched in enzymes characteristic of surface membranes and diminished in enzymes characteristic of intracellular organelles. Sialic acid content was also increased. SDS gel electrophoresis revealed the presence of a 45,000 molecular weight band, which may be actin. The preparation transported serine, glycine, and alpha-aminoisobutyric acid by a temperature-dependent, saturable process.

Alkaline Phosphatase↗
PubMedDate not supplied

Glycine uptake by microvillous and basal plasma membrane vesicles from term human placentae.

Like most amino acids, glycine is present in higher concentrations in the fetus than in the mother. Unlike most amino acids, animal studies suggest fetal concentrations of glycine are minimally in excess of those required for protein synthesis. Abnormal glycine utilization has also been demonstrated in small-for-gestational age human fetuses. The mechanism(s) of glycine uptake in the human placenta are unknown. In other mammalian cells glycine is a substrate for the A, ASC and Gly amino acid transport systems. In this study human placental glycine uptake was characterized using microvillous and basal plasma membrane vesicles each prepared from the same placenta. In both membranes glycine uptake was mediated predominantly by the sodium-dependent A system. Competitive inhibition studies suggest that in microvillous vesicles the small percentage of sodium-dependent glycine uptake not inhibited by methylaminoisobutyric acid (MeAIB) shares a transport system with glycine methyl ester and sarcosine, substrates of the Gly system in other tissues. In addition there are mediated sodium-independent and non-selective transport mechanisms in both plasma membranes. If fetal glycine availability is primarily contingent upon the common and highly regulated A system, glycine must compete with many other substrates potentially resulting in marginal fetal reserves, abnormal utilization and impaired growth.

Cell Membrane↗