Biomedical subjects

S S Miller

Publications and source records attributed to S S Miller.

At least 19 recordsLinked to original sources

PubMed1999

NADH-glutamate synthase in alfalfa root nodules. Genetic regulation and cellular expression.

NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary nitrogen assimilation in alfalfa (Medicago sativa L.) root nodules. Here we report that in alfalfa, a single gene, probably with multiple alleles, encodes for NADH-GOGAT. In situ hybridizations were performed to assess the location of NADH-GOGAT transcript in alfalfa root nodules. In wild-type cv Saranac nodules the NADH-GOGAT gene is predominantly expressed in infected cells. Nodules devoid of bacteroids (empty) induced by Sinorhizobium meliloti 7154 had no NADH-GOGAT transcript detectable by in situ hybridization, suggesting that the presence of the bacteroid may be important for NADH-GOGAT expression. The pattern of expression of NADH-GOGAT shifted during root nodule development. Until d 9 after planting, all infected cells appeared to express NADH-GOGAT. By d 19, a gradient of expression from high in the early symbiotic zone to low in the late symbiotic zone was observed. In 33-d-old nodules expression was seen in only a few cell layers in the early symbiotic zone. This pattern of expression was also observed for the nifH transcript but not for leghemoglobin. The promoter of NADH-GOGAT was evaluated in transgenic alfalfa plants carrying chimeric beta-glucuronidase promoter fusions. The results suggest that there are at least four regulatory elements. The region responsible for expression in the infected cell zone contains an 88-bp direct repeat.

Amino Acid Oxidoreductases↗

PubMed1998

Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule-enhanced MDH.

Malate dehydrogenase (MDH) catalyzes the readily reversible reaction of oxaloacetate reversible malate using either NADH or NADPH as a reductant. In plants, the enzyme is important in providing malate for C4 metabolism, pH balance, stomatal and pulvinal movement, respiration, beta-oxidation of fatty acids, and legume root nodule functioning. Due to its diverse roles the enzyme occurs as numerous isozymes in various organelles. While antibodies have been produced and cDNAs characterized for plant mitochondrial, glyoxysomal, and chloroplast forms of MDH, little is known of other forms. Here we report the cloning and characterization of cDNAs encoding five different forms of alfalfa MDH, including a plant cytosolic MDH (cMDH) and a unique novel nodule-enhanced MDH (neMDH). Phylogenetic analyses show that neMDH is related to mitochondrial and glyoxysomal MDHs, but diverge from these forms early in land plant evolution. Four of the five forms could effectively complement an E. coli Mdh- mutant. RNA and protein blots show that neMDH is most highly expressed in effective root nodules. Immunoprecipitation experiments show that antibodies produced to cMDH and neMDH are immunologically distinct and that the neMDH form comprises the major form of total MDH activity and protein in root nodules. Kinetic analysis showed that neMDH has a turnover rate and specificity constant that can account for the extraordinarily high synthesis of malate in nodules.

Amino Acid Sequence↗

PubMed1998

Multicenter study of oxygen-insensitive handheld glucose point-of-care testing in critical care/hospital/ambulatory patients in the United States and Canada.

OBJECTIVES: Existing handheld glucose meters are glucose oxidase (GO)-based. Oxygen side reactions can introduce oxygen dependency, increase potential error, and limit clinical use. Our primary objectives were to: a) introduce a new glucose dehydrogenase (GD)-based electrochemical biosensor for point-of-care testing; b) determine the oxygen-sensitivity of GO- and GD-based electrochemical biosensor test strips; and c) evaluate the clinical performance of the new GD-based glucose meter system in critical care/hospital/ambulatory patients. DESIGN: Multicenter study sites compared glucose levels determined with GD-based biosensors to glucose levels determined in whole blood with a perchloric acid deproteinization hexokinase reference method. One site also studied GO-based biosensors and venous plasma glucose measured with a chemistry analyzer. Biosensor test strips were used with a handheld glucose monitoring system. Bench and clinical oxygen sensitivity, hematocrit effect, and precision were evaluated. SETTING: The study was performed at eight U.S. medical centers and one Canadian medical center. PATIENTS: There were 1,248 patients. RESULTS: The GO-based biosensor was oxygen-sensitive. The new GD-based biosensor was oxygen-insensitive. GD-based biosensor performance was acceptable: 2,104 (96.1%) of 2,189 glucose meter measurements were within +/-15 mg/dL (+/-0.83 mmol/L) for glucose levels of < or = 100 mg/dL (< or = 5.55 mmol/L) or within +/-15% for glucose levels of > 100 mg/dL, compared with the whole-blood reference method results. With the GD-based biosensor, the percentages of glucose measurements that were not within the error tolerance were comparable for different specimen types and clinical groups. Bracket predictive values were acceptable for glucose levels used in therapeutic management. CONCLUSIONS: The performance of GD-based, oxygen-insensitive, handheld glucose testing was technically suitable for arterial specimens in critical care patients, cord blood and heelstick specimens in neonates, and capillary and venous specimens in other patients. Multicenter findings benchmark the performance of bedside glucose testing devices. With the new +/-15 mg/dL --> 100 mg/dL --> +/-15% accuracy criterion, point-of-care systems for handheld glucose testing should score 95% (or better), as compared with the recommended reference method. Physiologic changes, preanalytical factors, confounding variables, and treatment goals must be taken into consideration when interpreting glucose results, especially in critically ill patients, for whom arterial blood glucose measurements will reflect systemic glucose levels.

Adult↗

PubMed1998

Pseudomonas aeruginosa induces changes in fluid transport across airway surface epithelia.

Fluid transport across cultures of bovine tracheal epithelium was measured with a capacitance probe technique. Baseline fluid absorption (Jv) across bovine cells of 3.2 microliter. cm-2. h-1 was inhibited by approximately 78% after 1 h of exposure to suspensions of Pseudomonas aeruginosa, with a concomitant decrease in transepithelial potential (TEP) and increase in transepithelial resistance (Rt). Effects of P. aeruginosa were blocked by amiloride, which decreased Jv by 112% from baseline of 2.35 +/- 1.25 microliter. cm-2. h-1, increased Rt by 101% from baseline of 610 +/- 257 Omega. cm2, and decreased TEP by 91% from baseline of -55 +/- 18.5 mV. Microelectrode studies suggested that effects of P. aeruginosa on amiloride-sensitive Na absorption were due in part to a block of basolateral membrane K channels. In the presence of Cl transport inhibitors [5-nitro-2-(3-phenylpropylamino)-benzoic acid, H2-DIDS, and bumetanide], P. aeruginosa induced a fluid secretion of approximately 2.5 +/- 0.4 microliter. cm-2. h-1 and decreased Rt without changing TEP. However, these changes were abolished when the transport inhibitors were used in a medium in which Cl was replaced by an impermeant organic anion. Filtrates of P. aeruginosa suspensions had no effect on Jv, TEP, or Rt. Mutants lacking exotoxin A or rhamnolipids or with defective lipopolysaccharide still inhibited fluid absorption and altered bioelectrical properties. By contrast, mutations in the rpoN gene encoding a sigma factor of RNA polymerase abolished actions of P. aeruginosa. In vivo, changes in transepithelial salt and water transport induced by P. aeruginosa may alter viscosity and ionic composition of airway secretions so as to foster further bacterial colonization.

ADP Ribose Transferases↗

PubMed1998

cAMP-dependent absorption of chloride across airway epithelium.

Elevated levels of Na and Cl in airway surface liquid may play a major role in the airway pathology of cystic fibrosis (CF) (J. J. Smith, S. M. Travis, E. P. Greenberg, and M. J. Welsh. Cell 85: 229-236, 1996) and could be caused by block of transcellular Cl absorption due to lack of a functional CF transmembrane conductance regulator (CFTR). To test for transcellular absorption of Cl across non-CF epithelium, we studied how fluid absorption was affected by the opening and closing of Cl channels. Forskolin (an activator of CFTR) tripled fluid absorption across primary cultures of bovine tracheal epithelium but had no effect on human cells. However, in both species, fluid absorption was markedly inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, a blocker of CFTR. Microelectrode studies suggested that the magnitude of the absorptive response to forskolin in bovine cells depended on the size of an inwardly directed electrochemical driving force for Cl movement across the apical membrane. Patch-clamp measurements of bovine cells revealed CFTR in the apical membrane and a cAMP-activated, inwardly rectifying Cl channel in the basolateral membrane. We conclude that a significant fraction of absorbed Cl passes transcellularly in bovine tracheal epithelial cultures, with CFTR as the path of entry in the apical membrane and a novel cAMP-activated Cl channel as the exit route in the basolateral membrane. Our data further indicate that a similar pathway may exist in non-CF human tracheal epithelium.

Absorption↗

PubMed1998

Ultraspatially-resolved synchrotron infrared microspectroscopy of plant tissue in situ.

Routine use of 6 microm or 12 microm apertures with synchrotron microspectroscopy provide good spectra without excessive co-addition of scans. 100% mapping by stepping in pixel sized increments reveals chemical heterogeneity within cellular dimensions. The brightness of the synchrotron source and the absence of thermal noise compared to a conventional thermal (globar) source yields favorable signal-to-noise operation. The nondivergent characteristics of the source result in minimal loss of radiation at the aperture, hence, spatial resolution approaches the diffraction limit. Details of cellular dimensions are then localized within any maps produced and individual spectra obtained from adjacent pixels clearly shows the striking difference in chemistry even within a microscopic vicinity. In this report the mapping of plant tissue with the synchrotron is contrasted to previous lower spatial resolution mapping experiments done with the globar on similar materials using interpolation between separated sampling spots and larger apertures.

Absorption↗

PubMed1997

Fluid transport across cultures of human tracheal glands is altered in cystic fibrosis.

1. There is evidence that defective submucosal gland secretion contributes to the airway pathology of cystic fibrosis (CF). Using a capacitance probe technique, we have compared fluid transport across submucosal gland cultures from individuals with and without CF. 2. Under baseline conditions, approximately 60% of non-CF cultures secreted fluid; the rest absorbed. In secreting tissues, amiloride increased secretion, whereas in absorbing tissues it reduced or reversed absorption. 5-Nitro-2(3-phenylpropylamino)-benzoate (NPPB) a blocker of the CF transmembrane conductance regulator (CFTR), converted secretion to absorption. Thus, the direction and magnitude of baseline fluid movement depended on a balance between active absorption of Na+ and cAMP-dependent secretion of Cl-. 3. 8-(4-Chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP), methacholine and luminal uridine 5'-triphosphate (UTP) all induced or increased fluid secretion across non-CF cultures. Results with NPPB and with 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS), a blocker of Ca(2+)-activated Cl- channels, suggested that fluid secretion induced by CPT-cAMP was mediated primarily by CFTR; UTP acted entirely via Ca(2+)-activated Cl- channels, and methacholine activated both pathways. 4. All CF cultures showed baseline fluid absorption, which was abolished by amiloride. 5. CF cultures showed a normal secretory response to UTP, a reduced response to methacholine, and no response to CPT-cAMP. 6. Thus, the absorptive processes of airway glands are retained in CF, but the cAMP-dependent secretory process is lost. This would markedly reduce the water content of gland secretions. The resulting change in viscosity would contribute to the accumulation of airway mucus which is characteristic of this disease.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

Extracellular ATP activates calcium signaling, ion, and fluid transport in retinal pigment epithelium.

The presence of receptors for ATP has not been established in any native preparation of retinal neurons or glia. In the present study, we used conventional electrophysiological and [Ca2+]in fluorescence imaging techniques to investigate the effects of ATP added to Ringer's solution perfusing the retinal-facing (apical) membrane of freshly isolated monolayers of bovine retinal pigment epithelium (RPE). ATP (or UTP) produced large, biphasic voltage and resistance changes with a Kd of approximately 5 microM for ATP and approximately 1 microM for UTP. Electrical and pharmacological evidence indicates that the first and second phases of the response are attributable to an increase in basolateral membrane Cl conductance and a decrease in apical membrane K conductance, respectively. The ATP-induced responses were not affected by adenosine, but were reduced by the P2-purinoceptor blocker suramin. ATP also produced a large, transient increase in [Ca2+]in that was blocked by cyclopiazonic acid, an inhibitor of endoplasmic reticulum Ca2+-ATPases. The calcium buffer BAPTA attenuated the voltage effects of ATP. We also found that apical DIDS significantly inhibited the ATP-evoked [Ca2+]in and electrical responses, suggesting that DIDS blocked the purinoceptor. Measurements of fluid movement across the RPE using the capacitance probe technique demonstrated a significant increase in fluid absorption by apical UTP. These data indicate the presence of metabotropic P2Y/P2U-purinoceptors at the RPE apical membrane and implicate extracellular ATP in vivo as a retinal signaling molecule that could help regulate the hydration and chemical composition of the subretinal space.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

Resident learning and knowledge retention from resident-prepared chest radiology conferences.

RATIONALE AND OBJECTIVES: The authors assessed resident learning and retention of knowledge from resident-prepared chest radiology conferences. MATERIALS AND METHODS: Radiology residents presented five chest conferences to their peers during a 5-month interval; the conferences were modeled on a case presentation format. Tests were given 5 minutes before each conference (pretest) and immediately after each conference (posttest). The tests were readministered as a final examination 6 months later, at which time the residents were asked to evaluate the conference format. RESULTS: Conference attendance ranged from six to 11 residents. Mean posttest scores were statistically significantly higher than mean pretest scores (P < .0001). Six-month retention scores were higher than pretest scores (P < .05) but lower than posttest scores (P < .05). On a scale of 1-6, with 1 representing strongly disagree and 6 strongly agree, residents strongly agreed that the conferences provided an excellent learning experience (mean score, 5.27). CONCLUSION: Resident-prepared conferences are effective for teaching residents chest radiology. Resident testing at 6 months demonstrated retention of knowledge above pretest levels but lower than posttest levels.

Analysis of Variance↗

PubMed1997

Nitrogen assimilation in alfalfa: isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves.

Asparagine, the primary assimilation product from N2 fixation in temperate legumes and the predominant nitrogen transport product in many plant species, is synthesized via asparagine synthetase (AS; EC 6.3.5.4). Here, we report the isolation and characterization of a cDNA and a gene encoding the nodule-enhanced form of AS from alfalfa. The AS gene is comprised of 13 exons separated by 12 introns. The 5' flanking region of the AS gene confers nodule-enhanced reporter gene activity in transformed alfalfa. This region also confers enhanced reporter gene activity in dark-treated leaves. These results indicate that the 5' upstream region of the AS gene contains elements that affect expression in root nodules and leaves. Both AS mRNA and enzyme activity increased approximately 10- to 20-fold during the development of effective nodules. Ineffective nodules have strikingly reduced amounts of AS transcript. Alfalfa leaves have quite low levels of AS mRNA and protein; however, exposure to darkness resulted in a considerable increase in both. In situ hybridization with effective nodules and beta-glucuronidase staining of nodules from transgenic plants showed that AS is expressed in both infected and uninfected cells of the nodule symbiotic zone and in the nodule parenchyma. RNA gel blot analysis and in situ hybridization results are consistent with the hypothesis that initial AS expression in nodules is independent of nitrogenase activity.

Adaptation, Physiological↗

PubMed1997

Apical and basolateral membrane mechanisms that regulate pHi in bovine retinal pigment epithelium.

pH regulation was studied in fresh explant bovine retinal pigment epithelium-choroid using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and intracellular microelectrodes. Acid recovery was HCO3 dependent, inhibited by apical amiloride and apical or basal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and required apical and basal Na. Alkali recovery was HCO3 dependent and inhibitable by apical or basal DIDS. Three apical and two basolateral transporters were identified. Four contribute to acid extrusion, i.e., apical Na/H exchange, apical H-lactate cotransport, and apical Na-HCO3 cotransport and basolateral Na-HCO3 cotransport. At least two contribute to alkali extrusion, i.e., apical Na-HCO3 cotransport and a basolateral HCO3-dependent, DIDS-inhibitable mechanism, possibly Na-HCO3 cotransport, Cl/HCO3 exchange, or both. The apical Na-HCO3 cotransporter is electrogenic, carrying net negative charge inward. Basal Cl removal or addition of basal HCO3 caused HCO3- and Cl-dependent alkalinizations, respectively. Apical DIDS increased both responses. These cytosolic pH (pHi) regulatory mechanisms are so tightly coupled that changes in pHi can only occur after two or more of them are inhibited. In addition, these mechanisms help provide pathways for transport of Na and HCO3 across the retinal pigment epithelium between the blood and the distal retina.

Acids↗

PubMed1996

A new reusable instrument designed for simple and secure knot tying in laparoscopic surgery.

This new instrument is a metal cannula designed for use in creating controlled intracorporeal knots similar to instrument ties used in open surgery. It can therefore be used in a number of situations in laparoscopic surgery for ligation in continuity-for example, of the undivided cystic duct or of a vascular pedicle or for ligation of the cut end of a pedicle held in artery forceps. The cannula is designed to be loaded with 0 suture material and has a tip that readily penetrates the abdominal wall through a 3-mm skin incision, so it can be introduced either directly or through an introducer sleeve in a laparoscopic port. A curved dissecting forceps is used for knot formation. The knot is tightened by pushing the cannula with the other hand, and further throws are applied. The tip is profiled so that the knot, which is formed is pushed down into position without slippage or damage to the suture material.

Catheterization↗

PubMed1996

The anterior abdominal wall in laparoscopic procedures and limitations of laparoscopic simulators.

BACKGROUND: Maneuvers involving two-handed techniques such as intracorporeal suturing and knot-tying during laparoscopy are intrinsically more difficult than ones performed during open surgery. METHODS: The use of simulators to practice and teach specialized techniques is established. However, simulators vary greatly, and few, as yet, represent the abdominal wall well. RESULTS: This study has shown that the working angle between instruments during laparoscopic cholecystectomy is 78 degrees, and this can be increased to 117 degrees by moving the instruments laterally. In contrast, the working angle in four trainers assessed was never greater than 77 degrees. CONCLUSIONS: This suggests that some maneuvers may be more difficult in trainers than at surgery. This has implications for training and the further development of more realistic simulators.

Abdominal Muscles↗

PubMed1996

Cyclic responding by pigeons on the peak timing procedure.

The present experiment examined whether discrimination learning shapes the single-peaked response distributions usually obtained with the peak procedure. Two sources of learning in pigeons were disclosed: learning to respond near the time of reinforcement on fixed interval (FI) trials and learning to withhold responding once the FI duration had elapsed on peak interval (PI) trials. Pigeons also produced a highly unexpected second peak in responding on nonreinforced PI trials at 3 times the FI duration. Follow-up experiments showed that a 1:4 FI:PI duration ratio supported double peaks, but only 1 peak was obtained with a 1:8 FI:PI duration ratio. Finally, 4 peaks could be observed on extra-long PI trials under a 1:4:8 FI:PI:PI ratio procedure. The multiple-peaked response distributions are an unprecedented finding that present a major challenge to any theory of time perception.

Animals↗

PubMed1996

Nonsteroidal anti-inflammatory drugs alter chloride and fluid transport in bovine retinal pigment epithelium.

Nonsteroidal anti-inflammatory drugs (NSAIDs) were added to the solutions bathing the apical membrane of bovine retinal pigment epithelium (RPE)-choroid explants. For example, niflumic acid (100 microM) depolarized the basolateral membrane voltage (VB) by approximately 12 mV, increased transepithelial potential by 4.5 mV, decreased intracellular Cl activity by 13 mM, decreased transepithelial resistance by 17 omega.cm2, and increased the ratio of apical to basolateral membrane resistance nearly threefold. All of these changes are consistent with an increase in basolateral membrane Cl conductance. In addition, niflumic acid caused intracellular Ca concentration to decrease by 16 nM and fluid transport rate to increase by 1.5 microliters.cm-2.h-1. Flufenamic acid, which is structurally very similar to niflumic acid, had the opposite effects on membrane voltage and resistance. Basal application of the Cl channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or current clamping VB to the reversal potential for Cl practically abolished the niflumic acid response. The niflumic acid results suggest that certain NSAIDs can directly alter Cl conductance in the bovine RPE, apparently independently of cyclooxygenase inhibition.

Animals↗

PubMed1995

The delayed basolateral membrane hyperpolarization of the bovine retinal pigment epithelium: mechanism of generation.

1. Conventional and ion-selective double-barrelled microelectrodes were used in an in vitro preparation of bovine retinal pigment epithelium (RPE)-choroid to measure the changes in membrane voltage, resistance and intracellular Cl- activity (aCli) produced by small, physiological changes in extracellular potassium concentration ([K+]o). These apical [K+]o changes approximate those produced in the extracellular (subretinal) space between the photoreceptors and the RPE following transitions between light and dark. 2. Changing apical [K+]o from 5 to 2 mM in vitro elicited membrane voltage responses with three distinct phases. The first phase was generated by an apical membrane hyperpolarization, followed by a (delayed) basolateral membrane hyperpolarization (DBMH); the third phase was an apical membrane depolarization. The present experiments focus on the membrane and cellular mechanisms that generate phase 2 of the response, the DBMH. 3. The DBMH was abolished in the presence of apical bumetanide (100 microM); this response was completely restored after bumetanide removal. 4. Reducing apical [K+]o, adding apical bumetanide (500 mM), or removing apical Cl- decreased aCli by 25 +/- 6 (n = 8), 28 +/- 1 (n = 2) and 26 +/- 5 mM (n = 3), respectively; adding 100 microM apical bumetanide decreased aCli by 12 +/- 2 mM (n = 3). Adding apical bumetanide or removing apical bath Cl- hyperpolarized the basolateral membrane and decreased the apparent basolateral membrane conductance (GB). 5. DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid) blocked the RPE basolateral membrane Cl- conductance and inhibited the DBMH and the basolateral membrane hyperpolarization produced by apical bumetanide addition or by removal of apical Cl-o. The present results show that the DBMH is caused by delta[K]o-induced inhibition of the apical membrane Na(+)-K(+)-2Cl- cotransporter; the subsequent decrease in aCli generated a hyperpolarization at the basolateral membrane Cl- channel.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1995

Alfalfa NADH-dependent glutamate synthase: structure of the gene and importance in symbiotic N2 fixation.

Glutamate synthase (GOGAT), a key enzyme in ammonia (NH+4) assimilation, occurs as two forms in plants: a ferredoxin-dependent form (Fd-GOGAT) and an NADH-dependent form (NADH-GOGAT). These enzymes are encoded by distinct genes as evidenced by their cDNA and deduced amino acid sequences. This paper reports the isolation and characterization of a NADH-GOGAT gene from alfalfa (Medicago sativa L.), the first GOGAT gene to be isolated from a eukaryote. RNase protection and primer extension experiments map the transcription start site of NADH-GOGAT to nearly identical positions. The transcribed region of this gene, 12,214 bp, is comprised of 22 exons separated by 21 introns. The 2.7 kbp region 5' from the translation initiation site confers nodule-specific reporter gene activity when used in a chimeric beta-glucuronidase (GUS) construct and transformed into Lotus corniculatus and Medicago sativa. Both infected and uninfected cells display GUS activity. The abundance of NADH-GOGAT transcripts increases substantially in developing nodules of plants infected with effective rhizobia. However, this increase is not observed when nodules are induced by a variety of ineffective rhizobial strains. Thus, unlike many other plant genes involved in root nodule NH+4 assimilation, high levels of NADH-GOGAT expression are strictly associated with effective nodules indicating that NADH-GOGAT plays a central role in the functioning of effective root nodules. An alfalfa Fd-GOGAT PCR product showing greater than 85% identity to maize Fd-GOGAT was isolated and used to investigate the contribution of this enzyme to NH+4 assimilation in nodules. Fd-GOGAT mRNA was abundant in leaves and cotyledons but was not detected in alfalfa root nodules. Fd-GOGAT in alfalfa does not appear to play a significant role in symbiotic N2 fixation.

Amino Acid Sequence↗

PubMed1995

Identification and functional characterization of a dual GABA/taurine transporter in the bullfrog retinal pigment epithelium.

Intracellular microelectrodes, fluorescence imaging, and radiotracer flux techniques were used to investigate the physiological response of the retinal pigment epithelium (RPE) to the major retinal inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). GABA is released tonically in the dark by amphibian horizontal cells, but is not taken up by the nearby Müller cells. Addition of GABA to the apical bath produced voltage responses in the bullfrog RPE that were not blocked nor mimicked by any of the major GABA-receptor antagonists or agonists. Nipecotic acid, a substrate for GABA transport, inhibited the voltage effects of GABA. GABA and nipecotic acid also inhibited the voltage effects of taurine, suggesting that the previously characterized beta-alanine sensitive taurine carrier also takes up GABA. The voltage responses of GABA, taurine, nipecotic acid, and beta-alanine all showed first-order saturable kinetics with the following Km's: GABA (Km = 160 microM), beta-alanine (Km = 250 microM), nipecotic acid (Km = 420 microM), and taurine (Km = 850 microM). This low affinity GABA transporter is dependent on external Na, partially dependent on external Cl, and is stimulated in low [K]o, which approximates subretinal space [K]o during light onset. Apical GABA also produced a significant conductance increase at the basolateral membrane. These GABA-induced conductance changes were blocked by basal Ba2+, suggesting that GABA decreased basolateral membrane K conductance. In addition, the apical membrane Na/K ATPase was stimulated in the presence of GABA. A model for the interaction between the GABA transporter, the Na/K ATPase, and the basolateral membrane K conductance accounts for the electrical effects of GABA. Net apical-to-basal flux of [3H]-GABA was also observed in radioactive flux experiments. The present study shows that a high capacity GABA uptake mechanism with unique pharmacological properties is located at the RPE apical membrane and could play an important role in the removal of GABA from the subretinal space (SRS). This transporter could also coordinate the activities of GABA and taurine in the SRS after transitions between light and dark.

Animals↗