Lysine uptake by cloned hCAT-2B: comparison with hCAT-1 and with trophoblast surface membranes.

To study the cationic amino-acid transporter hCAT-2B of human placenta, total RNA was harvested from primary cultured trophoblast and from the BeWo choriocarcinoma cell line (b30 clone) and used for reverse transcription (RT) and polymerase chain reaction (PCR). Primers based on published sequences identified expression of mRNA for hCAT-2B. RT-PCR yielded a 2.06 kb hCAT-2B cDNA, which was cloned. hCAT-2B cRNA injection into Xenopus laevis oocytes stimulated saturable lysine uptake (Km approximately 125 mM). In the presence of Na+, uptake was completely inhibited by L-arginine but only partially by neutral amino acids. To compare directly the interaction of hCAT-1 and hCAT-2B with neutral amino acids and sodium, we examined the inhibition of these transporters by L-leucine and L-alanine over a wide concentration range. L-Alanine and L-leucine inhibit uptake by hCAT-2B substantially less completely than uptake by hCAT-1. The interaction of hCAT-2B resembles that of system y+ in the microvillous membrane of human placenta, while that of hCAT-1 is more comparable to that of system y+ in basal membrane. The identification and characterization of the various cationic amino-acid transporters of the human placenta have the potential to increase the understanding of the cellular mechanism of transplacental transfer.

Stable polarized expression of hCAT-1 in an epithelial cell line.

Our laboratory has recently identified and cloned three cationic amino-acid transporters of human placenta. We have now examined the plasma membrane domain localization and functional expression of one of these transporters, hCAT-1, in a polarized epithelial cell line (MDCK). To facilitate identification of expressed protein we first transferred the hCAT-1 cDNA to a vector with C-terminal green fluorescent protein (GFP). The resultant hCAT-1-CT-GFP fusion protein stimulated L-[3H] lysine uptake in Xenopus oocytes. In confluent monolayers of stably transfected cells grown on porous nitrocellulose filters, saturable uptake of L-[3H] lysine from the basolateral surface was stimulated 7-fold over that of untransfected cells. Concentration-dependence studies in Na+-free medium at pH 7.4 demonstrated a Km of approximately 68 +/- 13 microM and a Vmax of 970 +/-170 pmol/mg protein/min. Uptake from the apical plasma membrane surface was negligible in both transfected and untransfected cells. Consistent with these results, confocal microscopy of confluent monolayers of hCAT-1-CT-GFP-expressing cells revealed localization of the transporter solely on the basolateral domain of the cell. This is apparently the first report of a cultured polarized epithelial cell model for stable expression of a cationic amino-acid transporter. It has the potential to aid in the identification of targeting signals for transport protein localization.

Expression of neural markers in human umbilical cord blood.

A population of cells derived from human and rodent bone marrow has been shown by several groups of investigators to give rise to glia and neuron-like cells. Here we show that human umbilical cord blood cells treated with retinoic acid (RA) and nerve growth factor (NGF) exhibited a change in phenotype and expressed molecular markers usually associated with neurons and glia. Musashi-1 and beta-tubulin III, proteins found in early neuronal development, were expressed in the induced cord blood cells. Other molecules associated with neurons in the literature, such as glypican 4 and pleiotrophin mRNA, were detected using DNA microarray analysis and confirmed independently with reverse transcriptase polymerase chain reaction (RT-PCR). Glial fibrillary acidic protein (GFAP) and its mRNA were also detected in both the induced and untreated cord blood cells. Umbilical cord blood appears to be more versatile than previously known and may have therapeutic potential for neuronal replacement or gene delivery in neurodegenerative diseases, trauma, and genetic disorders.

Mitral valve repair: the Manipal alternative.

BACKGROUND AND AIM OF THE STUDY: An easily reproducible, rational and durable method of repairing the incompetent mitral valve, which does not require complex chordal procedures or the use of an expensive prosthesis and long-term anticoagulation, remains a desirable goal. Here, we describe such a method that has been developed at our institute. METHODS: The step-wise repair comprises: (i) preparation of a ring from a 3 x 110 mm strip of Dacron felt covered with untreated autologous pericardium; (ii) mitral commissurotomy and mobilization of the subvalvular apparatus, when required; (iii) infolding of the small portion of flail unsupported mitral leaflet, when present, by interrupted stitches; (iv) anchoring of the pre-prepared ring to the mitral annulus with interrupted horizontal mattress sutures, the sutures on the posterior annulus stopping short of the commissures by 12-15 mm and on the anterior annulus by 8-10 mm; (v) excision of the unanchored portions of the ring opposite the commissures, leaving behind 76-84 mm of the anchored parts; (vi) placement of two 'U-on-side' pericommissural annuloplasty sutures passed through the cut ends of the incomplete ring, then through the respective annulus, and finally emerging near the anterolateral and posteromedial commissures; and (vii) tying off the two pericommissural sutures over Teflon pledgets. RESULTS: Between January 1988 and December 1997, the technique was used to repair 107 mitral valves. Among 90 patients who had mitral valve repair alone or combined with tricuspid or aortic valve repair, only one hospital death occurred. One patient required reoperation due to an unacceptable degree of hemolysis. Among survivors followed up from one to >10 years, 80% were in NYHA functional class I, and 70% did not have clinical mitral regurgitation. CONCLUSION: This alternative technique of mitral valve repair is simple to perform, and relatively inexpensive. It provides gratifying results in acquired mitral valve disease, as well as in mitral valve prolapse subjects, and the repaired valve appears to function well, even after 10 years.

Tri-foliate pericardial patch repair of supra-valvular aortic stenosis.

Conventional aortoplasty procedures do not fully restore the normal anatomy in supra-valvular aortic stenosis (SVAS), which involves the sinus rim as well as the aortic cusps. A tri-sinus repair of this condition is proposed to restore the three intercommissural distances to normal and adequately replace the tissue loss of the sinuses, for a symmetrical reconstruction of the aortic root. Two patients, aged 3 and 11 years, with localized type of supra-valvular aortic stenosis were operated, in May 1994 and February 1996. The aortic stenosing ring was opened up at three points, by extending the incision into all the three sinuses, and the defect was repaired with in situ autologous pericardium, in a tri-foliate fashion. This repair achieved a symmetrical reconstruction of the aortic root and the systolic pressure gradient was completely abolished. Postoperative aortic root angiogram, in the older of the two patients, revealed a normal appearing aortic root. The patients have been followed-up for 51 months and 30 months respectively. Echocardiography showed competent aortic valves in both the patients without any systolic gradient across the aortic valve. A tri-sinus repair of the aorta in SVAS results in a symmetrical reconstruction of the aortic root by restoring the normal intercommissural distances of all the three cusps. It also abolishes the systolic pressure gradient. Autologous untreated pericardium lends itself easily for tailoring into a tri-foliate patch.

Tricuspid valve repair: a rational alternative.

BACKGROUND AND AIMS OF THE STUDY: The prosthetic ring annuloplasty and incompletely encircling suture techniques are effective methods of tricuspid valve repair when the problem is only annular dilatation, but not when organic tricuspid valve disease is present. A surgical technique of valve repair has been developed that is equally effective in correcting purely functional as well as organic valvular incompetence. METHODS: The Manipal method of repairing the incompetent tricuspid valve consists of three steps: (i) anteroseptal commissurotomy and asymmetric 'U-on-side' suture annuloplasty, to push the plane of coaptation of the anterior and septal leaflets into the right ventricle; (ii) a semicircular De Vega-type of plicating suture through the annulus, starting and ending just cephalad to the posteroseptal commissure and extending anticlockwise to a point just caudal to the meridian, to exclude the posterior leaflet; and (iii) tying the plicating suture after positioning a 3M Starr-Edward valve sizer across the tricuspid valve (in an adult), to ensure that the valve orifice is not excessively narrowed. RESULTS: Between July 1986 and January 1997, the Manipal method was used to repair 52 tricuspid valves, always combined with surgery for the mitral and/or aortic valve. Tricuspid stenosis of varying degree was present in 61% of cases. One of two hospital deaths was related to the repaired valve. Although the proportion of patients followed up fell progressively to 33% at 10 years, none of the patients either seen personally or who had replied to a postal questionnaire (78% of total patients) required reoperation for valve regurgitation or obstruction. No patient had more than mild tricuspid regurgitation clinically, even seven and 10 years after tricuspid valve repair surgery. CONCLUSION: This alternative method of tricuspid valve repair is simple to execute, is equally effective in correcting both pure tricuspid regurgitation and organic tricuspid valve disease, and appears to be extremely stable.

Identification of three cationic amino acid transporters in placental trophoblast: cloning, expression, and characterization of hCAT-1.

The concentrative transfer of amino acids from maternal to fetal blood is essential to fetal growth and metabolism. Cationic amino acids are transported across the placental microvillous and basal membranes by multiple pathways which act to mediate maternal/fetal transport. To identify the cationic amino acid transporters of human placenta, total RNA was harvested from cultured trophoblast and from the BeWo choriocarcinoma cell line, b30 clone, and used for reverse transcription (RT) and polymerase chain reaction (PCR). Primers based on published sequences identified expression of mRNAs for hCATs-1, -2B, and -4. RT-PCR yielded a 2.1 kb hCAT-1 cDNA which was cloned. hCAT-1 cRNA injection into Xenopus laevis oocytes stimulated saturable lysine uptake (K(m) approximately 100 microM). In the presence of Na(+), uptake was inhibited by leucine, homoserine, and alanine but not by valine and glutamate. These transport characteristics are comparable to those of system y(+) in placental basal membrane, but differ from those of the same system in microvillous membrane. The identification, cloning, and characterization of multiple human placental cationic amino acid transporters has the potential to facilitate molecular investigation of transport by the maternal- and fetal-facing membranes of placental trophoblast and increase understanding of the mechanism of transplacental amino acid transfer.

Pulmonary valve reconstruction in absent pulmonary valve syndrome: a new technique.

BACKGROUND: In patients with absent pulmonary valve syndrome, the relief of the pulmonary regurgitation at the time of primary repair improves both the early and late results. Though homograft and heterograft valves and conduits have been used for this purpose, both are not easily available and are known for late failure. Monocusp and bicuspid semilunar valves made out of pericardium have their own problems. Hence, a technique of reconstructing an autologous competent 3-cusp valve from the native tissues was developed. METHODS: Two posterolateral semilunar cusps were fashioned from the anterior wall of the main pulmonary artery. The anterior cusp was made from autologous pericardium stitched to the autologous pericardial patch used to widen the right ventricular outflow tract. RESULTS: This method of reconstruction was used in two patients aged 9 and 22 years, respectively. Visual assessment and passive testing after reconstruction revealed well functioning neopulmonary valves in both patients. The second patient, who had an unevenful hospital course, showed only mild pulmonary regurgitation at 5 years postreconstruction. CONCLUSIONS: As 2 of the 3 cusps are fashioned from the pulmonary arterial wall as a pedicled graft, we believe that they will retain their viability and grow with the pulmonary artery. Simultaneous reduction in the size of the pulmonary arteries will relieve bronchial compression when present. The anterior pericardial cusp, even if it eventually shrivels up, is unlikely to produce serious hemodynamic derangements.

Na+/Ca2+ exchange, Ca2+ binding, and electrogenic Ca2+ transport in plasma membranes of human placental syncytiotrophoblast.

To transfer a large amount of Ca2+ to the fetus, the basal (fetal-facing) plasma membrane (BPM) of human placenta must be equipped with various extrusion mechanisms. We studied one such mechanism, Na+/Ca2+ exchange, as well as related membrane potential effects and binding properties of the two membranes. Na+/Ca2+ exchange was present in BPM and absent in microvillous (maternal-facing) membrane. Uptake and efflux of Ca2+ in BPM were enhanced by Na+ when it was present on the opposite side of the membranes. Na(+)-gradient-dependent Ca2+ uptake was saturable with a Km of 19 microM and a Vmax of 0.8 nmol/min/mg. The Na+/Ca2+ exchange in BPM and the facilitated diffusion transporters in both BPM and microvillous membrane are electrogenic processes. Ca2+ binding in both BPM and microvillous membrane was affected by various monovalent cations and enhanced by Na+ more than by K+. In vivo, together with other sequestration mechanisms, Na+/Ca2+ exchange may play an important role in transsyncytial transfer and in regulating intracellular Ca2+, which is essential for a variety of physiologic mechanisms.

ATP independent calcium transport and binding by basal plasma membrane of human placenta.

The transport of large amounts of Ca2+ by the plasma membranes of human placental syncytiotrophoblast is essential to the mineralization of the growing fetal skeleton. We have investigated transport by the basal (fetal-facing) plasma membrane (BPM). Ca2+ was taken up by purified BPM vesicles in a time-dependent manner and equilibrium attained in approximately 60 min. The apparent equilibrium space was many fold higher than that determined using other substrates (e.g. leucine), suggesting that Ca2+ is concentrated or bound within the vesicles. The more rapid uptake and exit in the presence of A23187 indicates that membrane transport is rate limiting and that Ca2+ is internalized within the membrane space. The initial rate of uptake was approximately by measurement during the first 2 s of incubation. Concentration dependence data were fit to a Michaelis-Menten model with one saturable site and diffusion (Km = 12 microM; Vmax = 4 nmol/min/mg; KD = 39 nmol/min/mg/mM). Saturable Ca2+ binding (Kd = 16 microM; Bmax = 3.4 nmol/mg) was of lower capacity than previously observed for microvillous membrane.

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.