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[Biologic characterization of soft tissue sarcomes by positron emission tomography (initial results)].

Positron emission tomography (PET) with fluorine-18-fluorodeoxyglucose (FDG), carbon-11-labeled aminoisobutyric acid (AIB) and oxygen-15-labeled water (15O-labeled water) was evaluated for imaging soft tissue sarcomas (STS). Eleven pathologically confirmed STS were evaluated by PET using FDG, AIB and 15O-labeled water. All radiotracers showed an increased activity in viable tumor with a significant differentiation from normal tissue (muscle). An increased glucose metabolism in STS allowed a differentiation from normal tissue (muscle) with FDG. Furthermore, STS showed an increased AIB activity suggesting an increased "A-type" aminoacid transport. AIB therefore can be used for evaluation of STS. 15O-labeled water demonstrated an increased uptake with an overlap with blood vessel activity. These radiotracers supply information about biological properties of STS. Five patients with suspicious lesions in conventional radiodiagnostic but negative PET scans were free of local recurrence, diagnosed either by surgery or by follow-up. Two patients with PET findings suggesting local failure, ultimately demonstrated local recurrence. The differentiation of local recurrence from benign postoperative tissue alterations is encouraging and requires further evaluation.

Amino Acid Transport System A↗

Amino acid protection of cultured kidney tubule cells against calcium ionophore-induced lethal cell injury.

Treatment of two cultured renal tubule epithelial cell lines, MDCK and LLC-PK1, with ionomycin produced rapidly evolving models of lethal cell injury characterized by increases of cytosolic free calcium to the microM level within 15 minutes followed by lactate dehydrogenase release and failure to exclude vital dyes that began between 30 and 60 minutes and became extensive after 60 minutes. The pattern of injury was similar when the mitochondrial uncoupler, carbonyl cyanide-m-chlorophenylhydrazone, was added to ionomycin. Carbonyl cyanide-m-chlorophenylhydrazone alone produced severe ATP depletion but not lactate dehydrogenase release. Inclusion of glycine in the experimental medium at concentrations ranging from 0.25 mM to 5 mM did not affect the increases of cytosolic free calcium or ATP depletion but was protective against enzyme release and failure to exclude vital dyes for 180 minutes. Maximal protection was achieved at glycine concentrations between 1 and 5 mM. Several other small neutral amino acids including alanine, beta-alanine, L-serine, 1-aminocyclopropane-1-carboxylic acid, and alpha-aminoisobutyric acid also had protective effects but, glucose, pyruvate, glutamate, glutamine, leucine, valine, and taurine did not. These data indicate that potent protective effects of glycine and other small neutral amino acids previously shown in fresh tubule preparations are fully expressed in cultured tubule cells of diverse origin when appropriate acute injury models are used and the protective effects are sustained for long durations. The suitability of cultured cell lines for prolonged exposure studies will provide a powerful way of further exploring mechanisms of these effects.

Adenosine Triphosphate↗

Transport of potassium, amino acids, and glucose in cells transformed by Rous sarcoma virus.

Transport rates of a number of nutrients and ions have been surveyed in chicken embryo fibroblasts that were density inhibited, growing exponentially, or transformed by Rous sarcoma virus. All the transport systems examined displayed changes associated with changes in growth rate. The rate of ouabain-sensitive potassium transport declined in density-inhibited cells, and increased rapidly in response to serum stimulation. This transport system was regulated both by changes in the activity of the transporters and by the number of transporters in the cell membrane. The rate of transport of the amino acid analog alpha-aminoisobutyric acid declined when cells became density inhibited, but also showed alterations in regulation that were associated with malignant transformation. The rate of glucose transport displayed both growth state-related and transformation-specific changes. The increased rate of glucose transport seen in transformed cells is due to an increase in the number of glucose transporters in the cell membrane. Increased glucose transport is necessary for subsequent changes in glycolysis, and temporally precedes some of the changes in activity of glycolytic enzymes.

Amino Acids↗

Na+ + K+-ATPase activity and transport processes in toad corneal epithelium.

1. Ascorbic acid, diamide and N-ethylmaleimide inhibit Na+ + K+-ATPase activity in toad corneal epithelium. 2. Ascorbic acid, diamide and N-ethylmaleimide increase alpha-aminoisobutyric acid accumulation in this tissue. 3. The effects of these compounds on corneal amino acid and ion transport are not mediated through alterations in Na+ + K+-ATPase activity.

Aminoisobutyric Acids↗

Evidence that inhibition of muscle amino acid uptake during endotoxemia is not mediated by glucocorticoids.

Sepsis and endotoxemia are associated with increased muscle protein breakdown and inhibited amino acid uptake. Glucocorticoids are important for the regulation of muscle protein breakdown in catabolic conditions; in contrast, the role of glucocorticoids in the regulation of muscle amino acid transport during sepsis or endotoxemia is not known. The present study was designed to test the role of glucocorticoids in the regulation of muscle amino acid uptake during endotoxemia. Amino acid transport, determined as uptake of 3H-alpha-aminoisobutyric acid (AIB) by incubated soleus muscles in vitro, was reduced by approximately 40% 2 hours after intraperitoneal (IP) injection of 10 micrograms/kg endotoxin in rats. Administration of 5 mg/kg of the glucocorticoid receptor antagonist RU 38486 2 hours before endotoxin injection did not affect the inhibition of amino acid uptake. In vitro addition of plasma from endotoxemic rats to incubated rat soleus muscles inhibited amino acid uptake by approximately 30%. This effect of endotoxic plasma also was noted when muscles were from rats that had been treated with RU 38486 and when RU 38486 was present in the incubation medium. Results confirm previous reports of reduced muscle amino acid transport during endotoxemia and of the presence of a circulating factor that inhibits muscle amino acid uptake in this condition. Data suggest that inhibited muscle amino acid transport during endotoxemia is not regulated by glucocorticoids.

Aminoisobutyric Acids↗

Characteristics and regulation of proline transport in cultured glioblastoma cells.

L-Proline transport in C6 glioblastoma cells takes place mainly via a saturable Na(+)-dependent mechanism. The uptake process can be discriminated into two components, system A and system ASC. A minor proportion of L-proline transport is carried out by the ASC system, which appears to be constitutively expressed by the cell, but most is by system A which shows adaptive responses to amino acid deprivation and sensitivity to N-methyl-alpha-aminoisobutyric acid. The transport system is inhibited by proline derivatives, such as methyl and benzyl esters, and also hydroxyproline, and is stereospecific. Incubation of glioblastoma cells with phorbol 12-myristate 13-acetate led to concentration- and time-dependent decreases in L-proline transport. This effect could be mimicked by exogenous phospholipase C. Proline transport is significantly stimulated in the presence of Ca(2+)-mobilization agents and strongly inhibited in the absence of Ca2+. The present data suggest a complex regulation of L-proline transport by different kinases in glioblastoma cells.

Aminoisobutyric Acids↗

A mathematical model for membrane transport of amino acid and Na+ in vesicles.

A model with a carrier having sites for both amino acid and Na+ can account for AIB (alpha-aminoisobutyric acid) transport kinetics observed in membrane vesicles from SV3T3 (simian virus 40-tranformed Balb/c33 cells) and 3T3 (the parent cell line). The main feature of this cotransport model is that Na+ binding to carrier decreases the effective Km for AIB transport. Na+ transport kinetics observed in both vesicle systems can be described by passive (possibly facilitated) diffusion. The lag of Na+ transport across the membrane compared to that for AIB, coupled to the Na+-dependent decrease in the Km for AIB, accounts for the overshoot in intravesicular AIB observed for SV3T3 in the presence of an initial Na+ gradient. Extra-vesicular Na+ maintains a decrease in the Km for AIB influx before intra-vesicular Na+ has accumulated to balance it with a comparable decrease in the Km for AIB efflux. 3T3 vesicles display little overshoot, and this finding can be explained mostly by a lower carrier affinity for Na+.

Aminoisobutyric Acids↗

Role of the membrane potential in serum-stimulated uptake of amino acid in a diploid human fibroblast.

The Na+-dependent accumulation of alpha-aminoisobutyric acid (AIB), measured in normal growing and quiescent (serum-deprived) HSWP cells (human diploid fibroblast), was found to be twofold higher (AIB/in/AIBout = 20-25) under the normal growing conditions. Serum stimulation of quiescent cells increases their AIB concentrating capacity by approximately 70% within 1 hr. These observations suggest that the driving forces for AIB accumulation may be reversibly influenced by the serum concentration of the growth medium. Addition of valinomycin (Val) to cells preequilibrated with AIB causes an enhanced accumulation of AIB, suggesting that the membrane potential can serve as a driving force for AIB accumulation. After preequilibration with AIB in 6 mM K+, transfer to 94 mM K+ with Val results in a marked and rapid net loss of AIB. The effect of Val on the accumulation of AIB is greatest in quiescent cells, with the intracellular AIB concentrations reaching those seen both in Val-stimulated normal cells and in Val-stimulated serum-stimulated cells. By adjusting [K+]0, in the presence of Val, the membrane potential of growing cells can be matched to that of quiescent cells or vice versa. When this is done, the two accumulate AIB to the same extent. Hence the AIB accumulating capacity is characteristic of the membrane potential rather than of the growth state. In summary, these data suggest that the accumulation of AIB in HSWP cells is influenced by changes in membrane potential and that a serum-associated membrane hyperpolarization could be responsible for the increased capacity for AIB accumulation in serum-stimulated cells.

Aminoisobutyric Acids↗

Chain conformation in polyretropeptides III: design of a 3(10) helix using alpha,alpha-dialkylated amino acids and retropeptide bonds.

Computer simulations have been used to design a polypeptide with a 3(10) helix conformation. The study has been been performed taking advantage of the intrinsic helix forming tendency of alpha-Aminoisobutyric acid. In order to avoid the formation of the alpha helix, which is the other common helical conformation adopted by alpha-Aminoisobutyric acid-based peptides, retropeptide bonds have been included in the sequence. Thus, retropeptides are not able to form the intramolecular hydrogen bonding interactions characteristic of the alpha helix. The influences of both the peptide length and the solvent have been examined and compared with those of the polypeptide without retropeptide bonds.

Alkylation↗

Stereochemical control of peptide folding.

Stereochemically constrained amino acid residues that strongly favour specific backbone conformations may be used to nucleate and stabilize specific secondary structures in designed peptides. An overview of the use of alphaalpha-dialkyl amino acids in stabilizing helical structures in synthetic peptides is presented, with an emphasis on work carried out in the authors laboratory. Alpha-aminoisobutyric acid (Aib) and related achiral homologs facilitate stable helix formation in oligopeptides as exemplified by a large number of crystal structure determinations in the solid state. The ability to design conformationally rigid helical modules has been exploited in attempts to design structurally well characterized helix-linker helix, using potential nonhelical linking segments. Beta-hairpin design has been approached by exploiting the tendency of 'prime turns' to nucleate hairpin formation. The use of nucleating (D)Pro-Gly segments has resulted in the generation of several well characterized beta-hairpin structures, including the crystallographic observation of beta-hairpin in a synthetic apolar octapeptide. Extensions of this approach to three stranded beta-sheets and larger structures containing multiple (D)Pro-Gly segments appear readily possible.

Amino Acid Sequence↗

Maternal tobacco smoking and alterations in amino acid transport in human placenta: induction of transport systems.

The uptake of 14C-alpha-aminoisobutyric acid (AIB) by human placental villi from smokers and nonsmokers was measured. Washed placental villi of smokers exhibited higher uptake capacity for AIB than those of nonsmokers. The higher uptake capacity in smokers was due to increase in Vmax. Smoking increased Km and decreased affinity to AIB. This is possibly due to decreased fluidity of the plasma membrane of the villus during maternal smoking. These observations suggest that placenta utilizes its ability to increase the accumulation of amino acids during the adverse environmental conditions produced by maternal smoking.

Aminoisobutyric Acids↗

Characteristics of a neutral amino acid transport system (system A) in osteoblastic rat osteosarcoma cells.

Transport of alpha-aminoisobutyric acid (AIB) in the clonal, osteoblastic-like cell line, ROS 17/2, was characterized. AIB transport was time-, temperature- and Na+-dependent. Both ouabain and monensin inhibited AIB transport in these cells. AIB uptake followed Michaelis-Menten kinetics with an apparent Km = 0.57 mM and a Vmax = 4.07 nmol/30 min/plate. These characteristics are consistent with the presence of system A neutral amino acid transport in ROS 17/2 cells. Exposure of ROS 17/2 cells to either parathyroid hormone or dibutyryl cyclic AMP (db-cAMP), but not to dibutyryl cyclic GMP (db-cGMP), markedly stimulated AIB transport. This suggests that extracellular stimuli which enhance osteogenic responses in this cell type, coordinately upregulate system A transport.

Aminoisobutyric Acids↗

System A amino acid transport in cultured human tumor cells: implications for tumor imaging with PET.

The A system of amino acid transport is concentrative and thought to be a regulator of cell growth. The [11C]methyl alpha-aminoisobutyric acid (MeAIB) is prospectively an ideal tracer for transport measurements with PET, as it is not metabolized and concentrates in cells only via System A transport. We examined the factors governing [14C]MeAIB accumulation by cultured human erythroleukemic (K562) cells. Experiments were performed in growth medium and phosphate-buffered saline (PBS) +/- cycloheximide (an inhibitor of protein synthesis) on logarithmically growing cells, as well as cells that had reached a growth plateau. Both inward transport rate and net uptake of MeAIB were positively correlated with cell growth rate and showed a strong inverse relationship to amino acid supply. The observations are consistent with a body of evidence from animal tumor cells, and they suggest that the correlation between System A transport and tumor cell proliferation may be obscured in vivo by variations in amino acid supply. Thus, while [11C]MeAIB might be useful as a PET radiotracer of System A transport per se, this compound may be limited in its ability to provide measurements of tumor growth rate.

Amino Acid Transport Systems↗

Transmembrane potential and amino acid transport in rat hepatocytes in primary monolayer culture.

Transmembrane potential (Em) and alpha-aminoisobutyric acid (AIB) transport were measured in primary monolayer cultures of rat hepatocytes obtained from unoperated control rats and from rats 12 hr following partial hepatectomy. Measurements were performed 20-24 hr after plating the cells. The capacity of both kinds of cells to concentrate AIB depended upon extracellular sodium: however, the steady-state accumulation in regenerating cells was twice that of control cells. Transmembrane potentials, recorded with glass microelectrodes, were -13 +/- 0.6 mV and -27 +/- 1.6 mV in control and regenerating cells, respectively. Ouabain (1 mM) depolarized regenerating cell to -18 +/- 1.0 mV, but it had no effect on control cells. The initial rates of 1 mM AIB transport into control and regenerating cells were 1.2 +/- 0.1 and 3.1 +/- 0.1 nanomoles/mg protein x 4 min, respectively. Ouabain (1 mM) reduced the initial rate of AIB transport into regenerating cells to 2.7 +/- 0.1 nanomoles/mg protein x 4 min, but it had no effect on AIB transport into control cells. Glucagon (10(-7) M) added to control cells 12 hr before measurements hyperpolarized Em to -31 +/- 1.3 mV and increased AIB transport rate to 3.1 nanomoles/mg protein x 4 min. The results suggest a relationship between increases in Em and increases in AIB transport in rat hepatocytes. An electrogenic Na-K pump may be involved in both of these events.

Aminoisobutyric Acids↗

Carrageenin-induced arthritis. VI. Alterations in amino acid transport by articular cartilage in acute inflammatory arthritis.

The mechanism of transport of alanine and aminoisobutyric acid into chondrocytes in rabbit articular cartilage was shown to be mediated by transport systems similar to that described for other eukaryotic cells namely the A, ASC, and L systems. Three days after the initiation of an acute inflammatory arthritis by the intra-articular injection of carrageenin into one knee joint the rate of transport of both these amino acids was decreased. Although all three transport systems were depressed, it appeared that the A and ASC systems were partially susceptible to damage by the induced inflammation. The rate of amino acid transport by the affected cartilage had recovered by 28 days after carrageenin treatment. This depression in amino acid transport is discussed in relation to a decrease in general metabolic processes in chondrocytes as a consequence of inflammation.

Alanine↗

The effects of maternal protein deprivation on renal development and function in neonatal rats.

This study was undertaken to investigate the effects of pre- and/or postnatal maternal protein deprivation (PD) on renal functional development in the offspring. Pregnant rats were fed either a control (24% protein) or low (8 or 10%) protein diet during gestation and lactation. Progeny of these dams were cross-fostered at birth, yielding four experimental groups: pups born of control dams and nursed by PD or control dams and pups born of PD dams and nursed by control or PD dams. While the effects of prenatal PD on body and kidney growth were negligible, PD during the nursing period appeared to have a more profound effect on body and kidney weight. Renal transport functions, quantified in vitro, were differentially affected by these dietary manipulations. Renal transport capacity for organic acids and bases was depressed in pups nursed by PD dams. Maturation of the renal organic acid transport system was also delayed in these pups. alpha-Aminoisobutyric acid accumulation by renal cortical slices was enhanced only in 10-day old rats stressed by pre- and postnatal maternal PD. Renal gluconeogenic and ammoniagenic capacity was not impaired by these dietary manipulations. No differences in protein or water content of renal cortical slices were observed.

Aminoisobutyric Acids↗