Up-regulation of system A activity by hypertonic shock in CHO-K1 cells and an alanine-resistant mutant.
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Alanine↗
Biomedical subjects
A Felipe
Publications and source records attributed to A Felipe.
Differential expression of Isk mRNAs in mouse tissue during development and pregnancy.
The molecular isoform of the cDNA clone Isk present in the AT-1 atrial tumor cell line was characterized by molecular cloning of Isk cDNA. Since Isk mRNA was found in mouse heart, kidney, and uterus, a complete study of its expression during development in the heart and kidney was performed, in addition to its expression in the uterus during pregnancy. In the heart, Isk showed a 4-fold upregulation during the perinatal period followed by a 20-fold decrease between birth and the adult state. Furthermore, the two 0.9- and 3.4-kb transcripts were differentially regulated after birth. In the kidney, Isk progressively increased 10-fold, reaching steady-state adult values at 21 days. Isk mRNA levels in the uterus increased threefold at late pregnancy and decreased sixfold rapidly after birth. The Isk gene is differentially expressed during development in kidney and cardiac tissue, and both Isk transcripts appeared to be differentially regulated. Furthermore, the drastic changes in transcript levels before delivery and after birth suggest that Isk plays a significant role in myometrium during late pregnancy and delivery.
Amino Acid Sequence↗
K+ currents and K+ channel mRNA in cultured atrial cardiac myocytes (AT-1 cells).
Atrial tumor myocytes derived from transgenic mice (AT-1 cells) maintain a well-differentiated cardiac biochemical and histological phenotype. In addition, they beat spontaneously in culture and exhibit long action potentials whose repolarization resembles that observed in native mammalian myocytes. In this study, we identified the major depolarization-activated outward currents in AT-1 cells; also, the presence of mRNAs that encode outwardly conducting ion channels was determined by cloning from an AT-1 cDNA library or by Northern hybridization. Among K+ channel isoforms, Kv2.1, minK, and Kv1.4 were readily detected in tumors and at 1 day in culture. Their abundance remained relatively stable (twofold or less change) after 14 days. The major outward current in AT-1 cells is a delayed rectifier that displays prominent inward rectification, activates rapidly (eg, 182 +/- 27 milliseconds [mean +/- SEM] at + 20 mV, n = 12), exhibits biexponential deactivation kinetics, and is extremely sensitive to the methanesulfonanilide dofetilide (IC50, 12 nmol/L). These characteristics identify this current as IKr, a delayed rectifier observed only in cardiac cells. IKr in AT-1 cells displayed slow inactivation: dofetilide-sensitive deactivating tails were greater after 1-second than after 5-second pulses. When IKr was blocked by > or = 0.5 mumol/L dofetilide, time-independent current was usually recorded (50 of 65 experiments); rapidly inactivating (6 of 65) or slowly inactivating (9 of 65) outward currents were occasionally observed. We conclude that AT-1 cells express mRNAs encoding cardiac K+ channels and display a cardiac electrophysiological phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Contrast sensitivity of the visual system in speckle imagery.
The contrast sensitivity function (CSF) of the whole visual system is determined with the use of coherent diffuse illumination. This function provides supplementary data about the effect of speckle on the ability of the visual system to perceive the spatial information contained in an image. The results show that speckle not only prevents perception of the finest details (highest frequencies) but also reduces the visibility of lower frequencies (especially where contrast is low). The difference between the CSF's determined with and without speckle is quantitatively very important. And the ratio between the two CSF's is a measure of the retinal ability to perceive contrast in the presence of speckle noise. The influence of the pupil size and luminance level on the CSF with speckle is studied and compared with the influence of the same parameters on the classical CSF.
Contrast Sensitivity↗
Epidermal growth factor increases protein and messenger RNA expression levels of Ras GTPase activating protein.
The Ras GTPase activating protein (RasGAP) is a key regulatory enzyme in the Ras signaling pathway, which is crucial for growth factor-induced mitogenesis. In this study, it is shown that epidermal growth factor (EGF) increases RasGAP protein expression by 100-150% in NIH 3T3 cells, which overexpress the human EGF receptor, and mouse keratinocytes but does not increase RasGAP protein expression in A431 cells, where EGF does not have a mitogenic effect. In contrast, EGF does not affect the expression of other signal transduction SH2-containing proteins, such as phospholipase C gamma 1 and the p85 subunit of phosphatidylinositide 3-kinase. The growth factor-induced increase of RasGAP protein parallels an increase in RasGAP activity. EGF stimulates RasGAP protein synthesis and does not affect its degradation rate. The mechanism for RasGAP protein induction by EGF involves an increase in rasGAP mRNA levels. The growth factor-stimulated up-regulation of rasGAP is a delayed response, since the increase in the mRNA levels and protein synthesis rate begin after 3 h and reach maximal values between 9 and 24 h of growth factor treatment. EGF fails to increase rasGAP mRNA levels in the presence of cycloheximide, suggesting that this effect is dependent on de novo protein synthesis. However, cycloheximide alone is able to increase by 6-fold rasGAP mRNA expression. Since EGF does not modify rasGAP mRNA stability, the increase in rasGAP mRNA expression is likely due to an increase in transcription of the rasGAP gene.
Animals↗
Changes in the fatty acid composition of stored erythrocytes from sheep of different ages.
This study examined the profile in sheep erythrocyte fatty acids from animals of different ages during storage at 4 degrees C in a nutritive medium for up to 6 days. The changes found in the fatty acyl profile were a decrease (P < 0.01) in the percentage of arachidonic acid and an increase (P < 0.01) in the percentage of minor fatty acids (representing < 2% in each case; 20:2, 22:0 and 22:1) with respect to fresh erythrocytes in all age groups. However, the saturated/unsaturated ratios and unsaturated index started almost constant in all cases. The changes observed occurred after 24-48 h of storage, with significant increases (P < 0.01) in the fluorescence detected in the lipid extracts from stored erythrocytes during this period. The above findings suggest peroxidative damage and changes in the erythrocyte lipid membrane during storage.
Aging↗
Up-regulation of system A activity in the regenerating rat liver.
System A activity for neutral amino acid transport, measured as the MeAIB-sensitive Na(+)-dependent L-alanine uptake, is induced 6 h after partial hepatectomy in plasma membrane vesicles from rat livers. Other Na(+)-dependent transporters, like system ASC (MeAIB-insensitive Na(+)-dependent L-alanine transport) and the nucleoside carrier show similar inductions. Up-regulation of system A is not explained by changes in the dissipation rate of the Na+ transmembrane gradient, as deduced from uptake measurements performed in the presence of monensin. To determine whether induced system A shared any similarity with the activity found in hepatoma cell lines, we analyzed the N-ethylmaleimide (NEM) sensitivity of system A in both regenerating and control rat liver plasma membrane vesicles. NEM treatment was equally effective in inhibiting system A in both experimental groups. Thus, during the prereplicative phase of liver growth, a transport activity similar to basal system A is up-regulated in liver parenchymal cells, by a stable mechanism that does not involve changes in the Na+ transmembrane gradient.
Alanine↗
L-alanine uptake by rat liver parenchymal and haematopoietic cells during the perinatal period.
Alanine disposal by liver parenchymal and haematopoietic cells from 21-day fetuses, newborns and adult rats was studied. Preparations selectively enriched in either haematopoietic cells or hepatocytes were obtained by direct perfusion of fetal- and neonatal-rat livers. L-Alanine transport into liver parenchymal cells was best fitted to two Na(+)-dependent saturable systems. The high-affinity system showed a much higher activity (Vmax.) in hepatocytes from fetuses and newborns than in those from adult rats (2.4, 4.3 and 0.3 nmol/8 min per 10(6) cells for fetuses, newborns and adults respectively). Vmax. for the low-affinity component was slightly lower during the perinatal period than in the adult (about 30 nmol/8 min per 10(6) cells for hepatocytes from fetuses and newborns, versus 48 nmol/8 min per 10(6) cells for adult rat parenchymal cells). Haematopoietic cells from fetal-rat livers showed significant Na(+)-dependent L-alanine uptake which was completely abolished after birth. These results show that the transport systems involved in L-alanine uptake by liver parenchymal cells are fully developed before birth. This probably contributes to fulfilling the high requirement for neutral amino acids for protein synthesis during development. Haematopoietic cells may play an important role in liver amino acid metabolism during fetal life.
Alanine↗
Hyperosmolarity leads to an increase in derepressed system A activity in the renal epithelial cell line NBL-1.
Hyperosmolarity induced an increase in Na(+)-dependent L-alanine uptake in confluent monolayers of the established renal epithelial cell line NBL-1. This induction was attributable to system A and was only seen when the cells had been previously deprived of amino acids in the culture medium to derepress system A activity. It was additive to the adaptive regulation induction, and both were inhibited by cycloheximide. However, the hyperosmolarity effect was inhibited by colcemid (an inhibitor of microtubular function), but adaptive regulation was not. Otherwise, when cell monolayers were incubated in a control medium, basal Na(+)-dependent L-alanine uptake mediated by system B0 decreased. The results of this study show that: (i) system A activity was not induced by cell shrinkage and subsequent swelling due to extracellular hyperosmolarity when cells were incubated in control medium; (ii) previous expression of system A activity induced by amino acid starvation seems to be a prerequisite for further induction due to hyperosmolarity; and (iii) the effects of adaptive regulation and hyperosmotic stress are mediated by different mechanisms.
Alanine↗
Early induction of Na(+)-dependent uridine uptake in the regenerating rat liver.
Na(+)-dependent uridine transport into liver plasma membrane vesicles from partially hepatectomized and sham-operated rats was studied. Preparations purified 6 h after 70% hepatectomy exhibited an increased Vmax of uridine uptake (3.7 vs. 1.4 pmol/mg prot/3 s) without any change in Km (6 microM). Incubation of the vesicles in the presence of monensin decreased uridine uptake although the differences between both experimental groups remained identical. It is concluded that uridine transport is induced early after partial hepatectomy by a mechanism which does not involve changes in the transmembrane Na+ gradient. This is the first evidence in favor of modulation of nucleoside transport into liver cells.
Animals↗
Alanine uptake by liver of mid-lactating rats.
L-Alanine transport in liver plasma membrane vesicle preparations from fed virgin and 15-day-lactating rats was studied. Lactation was found to induce a decrease of the maximal rate (Vmax) of a high-capacity-low-affinity component of the Na(+)-dependent L-alanine uptake. However, a high-affinity-low-capacity agency was significantly induced in lactating-rat livers. L-Alanine uptake was differentially inhibited by other amino acids in those preparations from lactating rats, and showed different sensitivity to Li+ as a cosubstrate instead of Na+ and to inhibition by sulfhydryl modifying reagents (N-ethylmaleimide [NEM] and p-chloromercuribenzosulfonate [PCMBS]). All of these observations taken together suggest that system A is upregulated in lactating-rat livers, thus resulting in a different contribution of both agencies A and ASC to the total Na(+)-dependent alanine transport into liver plasma membrane vesicles. This was demonstrated using the analogue alpha-methyl-aminoisobutyric acid (MeAIB), a specific system A substrate. L-Alanine uptake rates, as calculated from plasma membrane enzyme marker recoveries, were also enhanced in the physiologic range of alanine concentrations in blood. Our results prove that the physiologic adaptation to lactation involves modulation of system A activity in the liver.
4-Chloromercuribenzenesulfonate↗
Influence of the contrast sensitivity function on the reaction time.
The reaction time (RT) vs spatial frequency (SF) curve is determined, using gratings from 1 to 40 c/deg, at seven different contrast levels between 0.95 and 0.02. The form of the RT/SF function: (a) replicated the inverse of the contrast sensitivity function (CSF) at near threshold contrast levels; (b) behaved differently at higher contrasts, exhibiting two branches at contrast close to 1. The interpretation is that there are two factors determining this function: (1) the transition from the operation of fast transient channels at low SF to the operation of slow sustained channels at high SF, the transition taking place within a narrow SF band close to 6 or 8 c/deg (depending on the subject) and (2) the contrast attenuation by the optical and neural transfer function, operating throughout the SF range. At high contrasts, the effect of the first factor can be clearly observed, because the effect of the second factor does not change with spatial frequency except in a region where the RT/SF function changes rapidly. At lower contrasts, however, the second factor becomes increasingly relevant while the first becomes less and less observable.
Contrast Sensitivity↗
Effect of color on contrast sensitivity with two different accommodative stimuli.
We studied the influence of color and accommodation on the contrast sensitivity function (CSF). At the same time, we measured the effect of axial chromatic aberration (ACA) on the CSF. The CSF's of two observers were determined using red, green, blue, and white light, at 5- and 0.5-m viewing distances. At 5 m the CSF's were measured with natural vision and also with lenses which compensated the ACA. Results show that the effect of ACA on the CSF is to reduce the sensitivity to blue with respect to the red. The difference in sensitivity between these two colors is between 50 and 150% and varies with the frequency and the subject. When the ACA is compensated the influence of the color on the CSF is very small, in our experiment, and this result is discussed in relation to the compensating effects of chromatic adaptation of the color mechanisms. In near vision, the CSF's were measured with natural vision and also with the lenses required to give the same accommodative stimulus for all colors. We discuss the effect of accommodation on the CSF. For both far and near the results are compared with the CSF with white light.
Accommodation, Ocular↗
Influence of cloned voltage-gated K+ channel expression on alanine transport, Rb+ uptake, and cell volume.
Voltage-gated K+ channels are involved in regulation of action potential duration and in setting the resting membrane potential in nerve and muscle. To determine the effects of voltage-gated K+ channel expression on processes not associated with electrically excitable cells, we studied cell volume, membrane potential, Na(+)-K(+)-ATPase activity, and alanine transport after the stable expression of the Kv1.4 and Kv1.5 human K+ channels in Ltk- mouse fibroblasts (L-cells). The fast-activating noninactivating Kv1.5 channel, but not the rapidly inactivating Kv1.4 channel, prevented dexamethasone-induced increases in intracellular volume and inhibited Na(+)-K(+)-ATPase activity by 25%, as measured by 86Rb+ uptake. Alanine transport, measured separately by systems A and ASC, was lower in Kv1.5-expressing cells, indicating that the expression of this channel modified the Na(+)-dependent amino acid transport of both systems. Expression of the Kv1.4 channel did not alter alanine transport relative to wild-type or sham-transfected cells. The changes specific to Kv1.5 expression may be related to the resting membrane potential induced by this channel (-30 mV) in contrast to that measured in wild-type sham-transfected, or Kv1.4-transfected cells (-2 to 0 mV). Blocking of the Kv1.5 channel by 60 microM quinidine negated the effects of Kv1.5 expression on intracellular volume, Na(+)-K(+)-ATPase, and Na(+)-dependent alanine transport. These results indicate that delayed rectifier channels such as Kv1.5 can play a key role in the control of cell membrane potential, cell volume, Na(+)-K(+)-ATPase activity, and electrogenic alanine transport across the plasma membrane of electrically unexcitable cells.
Alanine↗
Amino acid deprivation leads to the emergence of System A activity and the synthesis of a specific membrane glycoprotein in the bovine renal epithelial cell line NBL-1.
1. Amino acid deprivation of confluent monolayers of the bovine renal epithelial cell line NBL-1 causes a stimulation of Na(+)-dependent alanine transport. 2. This stimulation is mediated by a protein-synthesis-dependent induction of 2-(methylamino)isobutyric acid (methyl-AIB)-sensitive alanine transport activity (System A), which was not previously present in these cells. 3. Induction was prevented by the addition of methyl-AIB, alanine or glutamine. 4. Tunicamycin prevented the induction of alanine transport activity. 5. Induction of System A activity was accompanied by incorporation of [3H]mannose into a single membrane protein band of molecular mass 113-140 kDa. 6. These results are consistent with the possibility that induced System A activity in confluent NBL-1 cells is mediated by the synthesis of a 113-140 kDa membrane glycoprotein.
Alanine↗
Uridine transport in basolateral plasma membrane vesicles from rat liver.
The characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurements conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were KM 8.9 microM and Vmax 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na+ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rats and, thirdly, the permeant nature of the Na+ counterions determined uridine, transport rates (SCN- greater than NO3- greater than Cl- greater than SO4(2-)). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.
Animals↗
Changes in alanine and glutamine transport during rat red blood cell maturation.
Alanine and glutamine transport have been studied during red blood cell maturation in the rat. Kinetic parameters of Na(+)-dependent L-alanine transport were: Km 0.43 and 1.88 mM and Vmax 158 and 45 nmoles/ml ICW/min for reticulocytes and erythrocytes, respectively. During red cell maturation in the rat there is a loss of capacity and affinity of the system ASC for L-alanine transport. The values for Na(+)-dependent L-glutamine transport in reticulocytes were Km 0.51 mM and Vmax 157 nmoles/ml ICW/min. On the other hand, a total loss of L-glutamine transport mediated by both N and ASC systems is demonstrated in mature red cells. This seems to indicate that during rat red cell maturation the system N disappears. Furthermore, the system ASC specificity in mature cells changes, and glutamine enters the red cell by non-mediated diffusion processes.
Alanine↗
Control of neck nodes in squamous cell carcinoma of the head and neck by radiotherapy: prognostic factors.
313 patients with cervical metastases from a squamous carcinoma of the head and neck treated with radiotherapy, were studied by means of a multivariant analysis in order to determine the prognostic factors for cure. These were: lymph node response to irradiation (P = 0.0000), size of node (P = 0.0000), radiotherapy dose (P = 0.0037), condition of the primary (controlled vs non-controlled) (P = 0.0015), recurrent cervical metastases post-surgery (P = 0.0286).
Adolescent↗