Branched-chainα-amino acid chronic treatment: responses of plasmaα-keto-related compounds and ammonia when used in physical exercise performance

Summary To examine the effects of acute branched-chain-amino acids (BCAA) oral administration following chronic BCAA intake, a group of well trained young swimmers (n = 12) was submitted to a one month chronic BCAA treatment (0.2g/Kg body weight per die; Leu: Val: Ileu = 2:1:1) and a physical exercise test before and after this period of treatment was carried out. The exercise tests (60min swim) were performed in a high circulating BCAA level state which was obtained through oral BCAA administration (or placebo) just before the beginning of the exercise. The groups will be referred to as BCAA/before, BCAA/after, placebo/before, placebo/after. Blood and plasma (antecubital vein) samples were collected from the different groups at different times: on the morning of the day before the test (basal time, rest 0), the following day 30min after an acute administration (oral dose placebo or BCAA acute treatment: Leu 4.8g, Val 2.4g, Ileu 2.4g), just before the beginning of the exercise performance (time 0min, rest 1), at the end of the exercise (time 60min, EE) and during recovery (time 120min, Re). Plasma ammonia levels increased significantly from rest 1 to the end of the exercise in all subjects, but it was significantly higher in BCAA treated than in placebo subjects in both the before and after chronic treatment groups (BCAA/before: from 38 ± 7 to 204 ± 65mmol/l; placebo/before: from 36 ± 10 to 93 ± 29mmol/l; BCAA/after: from 36 ± 9 to 171 ± 43mmol/l; placebo/after: from 30 ± 6 to 65 ± 16mmol/l). Plasma ammonia level increments observed before a chronic one month BCAA treatment were significantly higher than after this treatment (p < 0.05). Plasma alanine was at all times of the test higher before the BCAA chronic treatment than after; this difference resulted significant at rest 0, rest 1 and recovery times (p < 0.05). After acute BCAA administration, plasma BCAA levels increased from 618 ± 52mmol/l to 1893 ± 284mmol/l (p < 0.05) from the onset of exercise and remained elevated throughout the test. Placebo and basal (rest 0) levels both before and after the chronic treatment did not demonstrate any significant differences. Plasma BCAA and BCKA levels, in the BCAA/before demonstrated significantly higher levels than placebo/before at rest 1 time (BCAA/before vs placebo/before: Leu 86 ± 27 vs 620 ± 97mmol/l; KIC 60 ± 3 vs 87 ± 5mmol/l, Ileu 51 ± 19 vs 359 ± 56mmol/l, KMV 26 ± 1 vs 43 ± 2mmol/l, Val 290 ± 79 vs 915 ± 133mmol/l, KIV 14 ± 1 vs 24 ± 2mmol/l). The levels after the chronic treatment maintained circa these differences in the two groups BCAA/after and placebo/after. The plasma BCAA as well as the BCKA levels of acutely treated athletes, in physical exercise, showed a different profile before and after the chronic treatment. The chronic treated BCAA/after group in fact depicted a decreasing BCKA level profile at the end of the exercise and during recovery; on the contrary, before the chronic treatments, acutely treated athletes demonstrated a tendency to increase these levels during recovery. These data seem to confirm that increased BCAA availability, before exercise, result in significantly greater plasma ammonia responses during exercise than does placebo administration; furthermore this increment is lower after chronic treatment. The interpretation of the ammonia data is difficult since the exercise type could have an influence on this phenomenon. The differences in the profile patterns of alanine, BCAA and BCKA levels seem to indicate that the chronic treatment brings about a state in which there is a better use of BCAA compounds as energy supply.Name and location where the investigations were carried out.     

Inhibition of brain energy metabolism by the alpha-keto acids accumulating in maple syrup urine disease

Neurological dysfunction is a common finding in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of brain damage in this disorder are poorly known. In the present study, we investigated the effect of the in vitro effect of the branched chain alpha-keto acids (BCKA) accumulating in MSUD on some parameters of energy metabolism in cerebral cortex of rats. [14CO(2)] production from [14C] acetate, glucose uptake and lactate release from glucose were evaluated by incubating cortical prisms from 30-day-old rats in Krebs-Ringer bicarbonate buffer, pH 7.4, in the absence (controls) or presence of 1-5 mM of alpha-ketoisocaproic acid (KIC), alpha-keto-beta-methylvaleric acid (KMV) or alpha-ketoisovaleric acid (KIV). All keto acids significantly reduced 14CO(2) production by around 40%, in contrast to lactate release and glucose utilization, which were significantly increased by the metabolites by around 42% in cortical prisms. Furthermore, the activity of the respiratory chain complex I-III was significantly inhibited by 60%, whereas the other activities of the electron transport chain, namely complexes II, II-III, III and IV, as well as succinate dehydrogenase were not affected by the keto acids. The results indicate that the major metabolites accumulating in MSUD compromise brain energy metabolism by blocking the respiratory chain. We presume that these findings may be of relevance to the understanding of the pathophysiology of the neurological dysfunction of MSUD patients.     

Evidence for a non-antioxidant,dose-dependent role of α -lipoic acid in caspase-3 and ERK2 activation in endothelial cells

Endothelial cell apoptosis contributes to atherosclerosis and may be exacerbated by oxidative stress. Results from clinical trials using antioxidant supplementation are equivocal and could be enhanced by antioxidants with additional non-antioxidant properties such as -lipoic acid and -tocopherol. The aim of this study was to investigate the effects of these antioxidants on cytoprotective pathways and endothelial apoptosis. Endothelial cells were incubated with -lipoic acid and -tocopherol, alone or in combination, prior to incubation with H₂O₂ or staurosporine. -lipoic acid pre-treatment alone increased caspase-3 activity in a dose-dependent manner. Both H₂O₂ and staurosporine increased DNA fragmentation and caspase-3 activity and pre-treatment of cells with -lipoic acid and/or -tocopherol failed to prevent stress-induced apoptosis. Neither antioxidant treatments nor apoptotic inducers alone altered expressions of Bcl-2, Bax, HSP70 or pERK1/2 or pJNK. -lipoic decreased pERK2 in staurosporine-treated cells in a dose-dependent manner. These findings indicate that pre-incubation with -lipoic acid and -tocopherol, alone or in combination, does not protect against oxidative- or non-oxidative-induced apoptosis in endothelial cells. Moreover, we have demonstrated a non-antioxidant, dose-dependent role of -lipoic acid in caspase-3 and ERK2 activation. These data provide an insight and indicate caution in the use of high doses of -lipoic acid as an antioxidant.     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

Effect of hyperprolinemia on acetylcholinesterase and butyrylcholinesterase activities in rat

Summary. We observed here that acute proline (Pro) administration provoked a decrease (32%) of acetylcholinesterase (AChE) activity in cerebral cortex and an increase (22%) of butyrylcholinesterase (BuChE) activity in the serum of 29-day-old rats. In contrast, chronic administration of Pro did not alter AChE or BuChE activities. Furthermore, pretreatment of rats with vitamins E and C combined or alone, N-nitro-L-arginine methyl ester or melatonin prevented the reduction of AChE activity caused by acute Pro administration, suggesting the participation of oxidative stress in such effects.     

Increase of Adenine Nucleotide Hydrolysis in Rat Hippocampal Slices after Seizures Induced by Quinolinic Acid

Quinolinic acid (QUIN), an endogenous convulsant compound, overstimulates the glutamatergic system stimulating N-methyl-D-aspartate receptors, enhancing glutamate release and inhibiting glutamate uptake. Glutamate releases the neuroprotector adenosine, which in turn reduces glutamate release and depresses the neuronal activity. Additionally, adenine nucleotides are an important source of adenosine, by action of ecto-nucleotidases. Here we evaluated the adenine nucleotide hydrolysis in hippocampal slices of adult rats in different times after seizures induced by QUIN. After 45 min, there was an increase of ATP and ADP hydrolysis. After 5 h, there was an increase of ATP, ADP and AMP hydrolysis. After 12 h, there was an increase only of ATP hydrolysis. After 24 h, all hydrolysis returned to control levels. As slice preparations maintain tissue integrity, this study indicates, more than previously observed with synaptosomal preparations, that the extracellular production of the neuroprotector adenosine may be involved in brain responses to seizures.     

A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins

A selective protonation strategy is described that uses [3-2H] 13C -ketoisovalerate to introduce (1H- methyl)-leucine and (1H- methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of 100 mg/L -ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] -ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-1 methyl)-isoleucine (>90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.     

Morphological alterations and induction of oxidative stress in glial cells caused by the branched-chain alpha-keto acids accumulating in maple syrup urine disease

Maple syrup urine disease (MSUD) is an inherited neurometabolic disorder biochemically characterized by the accumulation of the branched-chain alpha-keto acids (BCKA) alpha-ketoisocaproic (KIC), alpha-keto-beta-methylvaleric (KMV) and alpha-ketoisovaleric (KIV) and their respective branched-chain alpha-amino acids in body fluids and tissues. Affected MSUD patients have predominantly neurological features, including cerebral edema and atrophy whose pathophysiology is not well established. In the present study we investigated the effects of KIC, KMV and KIV on cell morphology, cytoskeleton reorganization, actin immunocontent and on various parameters of oxidative stress, namely total antioxidant reactivity (TAR), glutathione (GSH) and nitric oxide concentrations, and on the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in C6 glioma cells. We initially observed that C6 cultivated cells exposed for 3 h to the BCKA (1 and 10 mM) changed their usual rounded morphology to a fusiform or process-bearing cell appearance, while 24 h exposure to these organic acids elicited massive cell death. Rhodamine-labelled phalloidin analysis revealed that these organic acids induced reorganization of the actin cytoskeleton with no modifications on total actin content. It was also observed that 3h cell exposure to low doses of all BCKA (1 mM) resulted in a marked reduction of the non-enzymatic antioxidant defenses, as determined by TAR and GSH measurements. In addition, KIC provoked a reduced activity of SOD and GPx, whereas KMV caused a diminution of SOD activity. In contrast, CAT activity was not modified by the metabolites. Furthermore, nitric oxide production was significantly increased by all BCKA. Finally, we observed that the morphological features caused by BCKA on C6 cells were prevented by the use of the antioxidants GSH (1.0 mM), alpha-tocopherol (trolox; 10 microM) and Nomega-nitro-L-arginine methyl ester (L-NAME; 500 microM). These results strongly indicate that oxidative stress might be involved in the cell morphological alterations and death, as well as in the cytoskeletal reorganization elicited by the BCKA. It is presumed that these findings are possibly implicated in the neuropathological features observed in patients affected by MSUD.     

Rapid and Efficient Synthesis of Peptides Containing α,α-dialkylamino acids employing KOBt

Several Fmoc-,-dialkylamino acids and their acid chlorides have been prepared, isolated and characterised. The synthesis of peptides containing sterically hindered dialkylamino acids has been accomplished using acid chloride/KOBt in dichloromethane. The yields as well as the purity of the peptides were satisfactory.     

Tail-to-tail orientation of the Atlantic salmon alpha- and beta-globin genes

We report the cloning of a cDNA and two corresponding -globin genes of the Atlantic salmon (Salmo salar L.) as well as two genes for -globins. Nucleotide sequence analysis of the cDNA shows that the predicted -globin peptide comprises 148 amino acids with a calculated molecular mass of 16,127 Da and an overall amino acid similarity of 40–50% to higher vertebrates and 60–90% to fish sequences. The study of the genomic organization of - and -globin genes shows that, as is the case in Xenopus, the salmon genes are adjacent. Two sets of linked - and -globin genes were isolated and restriction-enzyme polymorphisms indicate that they belong to two distinct loci, possibly as a result of the salmon tetraploidy. In each locus the - and -globin genes are oriented 3 to 3 relative to each other with the RNA coding sequences located on opposite DNA strands. This is the first evidence for this type of arrangement found for globin genes. Moreover, while the linkage found in salmon and Xenopus supports the hypothesis of an initial tandem duplication of a globin ancestor gene, our results raise the question of the actual original orientation of the duplicated genes. Correspondence to: F. Gannon     

Ontogeny of adenosine 3′,5′-monophosphate metabolism in rabbit cerebral cortex

The effects of norepinephrine (NE), histamine (HIST), glutamate, and adenosine, singly and in combinations, on the accumulation of adenosine 3,5-monophosphate (cyclic AMP) in slices of rabbit cerebral cortex were examined using tissue from animals 4 days before to 38 days after birth. A response to NE became visible 2 days before birth and increased to - maximum at 7 days after birth before declining toward the small adult value during the second post-natal week. During this period NE was at least twice as efficacious as isoproterenol, and both - and -adrenergic antagonists had prominent inhibitory effects. Responses to HIST were already apparent 4 days before birth and increased in an irregular fashion thereafter, sometimes exceeding the adult value during the second post-natal week. The response to adenosine was not visible until birth and gradually increased toward the adult value during the entire period examined. Synergistic responses to various combinations of the three agents were first detected at 2 to 4 days before birth. The degree of synergism was larger during the neonatal period than that found in adult tissue; no synergism between HIST and adenosine persisted in the adult. During the first post-natal week, L-glutamate produced very large increases of cyclic AMP accumulation in the presence of either adenosine or histamine plus theophylline; smaller but substantial responses occurred in combination with NE. Responses to glutamate declined progressively after about the tenth post-natal day.     

Evaluation of two new coupling agents for incorporation of α,α-dialkylamino acids, such as α-methylalanine, in solid-phase peptide synthesis

Summary The introduction of solid-phase peptide synthesis (SPPS) has greatly facilitated the preparation of peptides containing proteinaceous amino acids. Less common, sterically hindered ,-dialkylamino acids, such as -methylalanine (MeA, aminoisobutyric acid, Aib), have proven a synthetic challenge for incorporation by this approach, especially when present in contiguous sequences. Solution protocols, utilizing highly reactive intermediates such as oxazalones, are generally used during the preparation of peptaibol antibiotics such as alamethicin, emerimicin, etc. which contain such contiguous sequences. Two recently developed coupling strategies (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, HATU, and Fmoc-protected amino acid fluorides) allow peptides comprising contiguous sequences of ,-dialkylamino acids to be prepared using SPPS. The present study evaluates the relative merits of these two methods on a set of difficult peptides containing oligo-MeA sequences.     

Receptor Interactions of β-N-Oxalyl-L-α,β-Diaminopropionic Acid,the Lathyrus sativus Putative Excitotoxin,with Synaptic Membranes

Direct evidence for the excitotoxicity of -N-oxalyl-L-,-diaminopropionic acid (ODAP), the Lathyrus sativus neurotoxin has been studied by examining the binding of chemically synthesized [2,3 ³H]ODAP ([³H]ODAP) to synaptic membranes. [³H]ODAP binding to membranes was mostly nonspecific, with only a very low specific binding (15–20% of the total binding) and was also not saturable. The low specific binding of [³H]ODAP remained unaltered under a variety of assay conditions. A low B_max of 3.2 ± 0.4 pmol/mg and K_d 0.2 ± 0.08 M could be discerned for the high affinity interactions under conditions wherein more than 80–90% of the binding was nonspecific. While ODAP could inhibit the binding of [³H]glutamate to chick synaptic membranes with a K_i of 10 ± 0.9 M, even L-DAP, a non neurotoxic amino acid was also equally effective in inhibiting the binding of [³H]glutamate. The very low specific binding of [³H]ODAP to synaptic membranes thus does not warrant considering its interactions at glutamate receptors as a significant event. The results thus suggest that the reported in vitro excitotoxic potential of ODAP may not reflect its true mechanism of neurotoxicity.     

Molecular cloning and characterization of a gibberellin-inducible, putative α-glucosidase gene from barley

A putative -glucosidase clone has been isolated from a cDNA library constructed from mRNA of barley aleurones treated with gibberellin A₃ (GA). The clone is 2752 bp in length and has an uninterrupted open reading frame encoding a polypeptide of 877 amino acids. A 680 amino acid region is 43% identical to human lysosomal -glucosidase and other glycosyl hydrolases. In isolated aleurones, the levels of the corresponding mRNA increase strongly after the application of GA, similar to the pattern exhibited by low-pI -amylase mRNA. High levels are also observed in the aleurone and scutellum after germination, while low levels are found in developing seeds. The genome contains a single form of this -glucosidase gene and two additional sequences that may be related genes or pseudogenes.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Glutamate Receptor Requirement for Neuronal Death from Anoxia–Reoxygenation: An in Vitro Model for Assessment of the Neuroprotective Effects of Estrogens

1.Previous studies demonstrated that estrogens, specifically 17-estradiol, the potent, naturally occurring estrogen, are neuroprotective in a variety of models including glutamate toxicity. The aim of the present study is twofold: (1) to assess the requirement for glutamate receptors in neuronal cell death associated with anoxia–reoxygenation in three cell types, SK-N-SH and HT-22 neuronal cell lines and primary rat cortical neuronal cultures, and (2) to evaluate the neuroprotective activity of both 17-estradiol and its weaker isomer, 17-estradiol, in both anoxia-reoxygenation and glutamate toxicity.2.SK-N-SH and HT-22 cell lines, both of which lack NMDA receptors as assessed by MK-801 binding assays, were resistant to both anoxia–reoxygenation and glutamate-induced cell death. In contrast, primary rat cortical neurons, which exhibit both NMDA and AMPA receptors, were sensitive to brief periods of exposure to anoxia–reoxygenation or glutamate. As such, there appears to be an obligatory requirement for NMDA and/or AMPA receptors in neuronal cell death resulting from brief periods of anoxia followed by reoxygenation.3.Using primary rat cortical neuronal cultures, we evaluated the neuroprotective activity of 17-estradiol (1.3 or 133 nM) and 17-estradiol (133 nM) in both anoxia–reoxygenation and excitotoxicity models of cell death. We found that the 133 nM but not the 1.3 nM dose of the potent estrogen, 17-estradiol, protected 58.0, 57.5, and 85.3% of the primary rat cortical neurons from anoxia–reoxygenation, glutamate, or AMPA toxicity, respectively, and the 133 nM dose of the weak estrogen, 17-estradiol, protected 74.6, 81.7, and 85.8% of cells from anoxia–reoxygenation, glutamate, or AMPA toxicity, respectively. These data demonstrate that pretreatment with estrogens can attenuate glutamate excitotoxicity and that this protection is independent of the ability of the steroid to bind the estrogen receptor.     

Chronic hypoxia in development selectively alters the activities of key enzymes of glucose oxidative metabolism in brain regions

The immature brain is more resistant to hypoxia/ischemia than the mature brain. Although chronic hypoxia can induce adaptive-changes on the developing brain, the mechanisms underlying such adaptive changes are poorly understood. To further elucidate some of the adaptive changes during postnatal hypoxia, we determined the activities of four enzymes of glucose oxidative metabolism in eight brain regions of hypoxic and normoxic rats. Litters of Sprague-Dawley rats were put into the hypoxic chamber (oxygen level maintained at 9.5%) with their dams starting on day 3 postnatal (P3). Age-matched normoxic rats were use as control animals. In P10 hypoxic rats, lactate dehydrogenase (LDH) activity in cerebral cortex, striatum, olfactory bulb, hippocampus, hypothalamus, pons and medulla, and cerebellum was significantly increased (by 100%–370%) compared to those in P10 normoxic rats. In P10 hypoxic rats, hexokinase (HK) activity in hypothalamus, hippocampus, olfactory bulb, midbrain, and cerebral cortex was significantly decreased (by 15%–30%). Neither -ketoglutarate dehydrogenase complex (KGDHC, which is believed to have an important role in the regulation of the tricarboxylic acid [TCA] cycle flux) nor citrate synthase (CS) activity was significantly decreased in the eight regions of P10 hypoxic rats compared to those in P10 normoxic rats. In P30 hypoxic rats, LDH activity was only increased in striatum (by 19%), whereas HK activity was only significantly decreased (by 30%) in this region. However, KGDHC activity was significantly decreased in olfactory bulb, hippocampus, hypothalamus, cerebral cortex, and cerebellum (by 20%–40%) in P30 hypoxic rats compared to those in P30 normoxic rats. Similarly, CS activity was decreased, but only in olfactory bulb, hypothalamus, and midbrain (by 9%–21%) in P30 hypoxic rats. Our results suggest that at least some of the mechanisms underlying the hypoxia-induced changes in activities of glycolytic enzymes implicate the upregulation of HIF-1. Moreover, our observation that chronic postnatal hypoxia induces differential effects on brain glycolytic and TCA cycle enzymes may have pathophysiological implications (e.g., decreased in energy metabolism) in childhood diseases (e.g., sudden infant death syndrome) in which hypoxia plays a role.     

Anthocyanin accumulation and PAL activity in a suspension culture of Daucus carota L.

Cells of Daucus carota grown in a liquid medium produced large amounts of cyanidin as the only flavonoid aglycon. After inoculation in fresh medium a maximum activity of phenylalanine ammonia lyase (PAL; EC 4.3.1.5) was observed within 24 h. L--aminooxy--phenylpropionic acid (L-AOPP), thought to be a competitive inhibitor of PAL, inhibited cyanidin accumulation up to 80%. In order to study the regulatory role of PAL, the effects of L-AOPP and t-cinnamic acid, the product of the deamination of phenylalanine, were investigated. Cinnamic acid, applied in vivo (10^-4 M), was not able to compensate for the inhibition of cyanidin production caused by L-AOPP (10^-4 M) in the same sample. Carrot cells treated with L-AOPP exhibited a super-induction of PAL already described for gherkin hypocotyls (Amrhein and Gerhardt 1979). This effect was not influenced by t-cinnamic acid. L-AOPP seems to be a very specific inhibitor since it affected neither growth nor soluble protein content, whereas t-cinnamic acid inhibited both. Investigations on the content of soluble amino acids in L-AOPP-treated cells revealed a specific accumulation of soluble phenylalanine, whereas treatment with t-cinnamic acid led to an increase of amino acids in general, thus indicating that the latter compound has a rather unspecific effect on cellular metabolism. In vitro studies with PAL isolated from Daucus carota revealed that L-AOPP inhibited the enzyme at very low doses (K _I=2.4·10^-9), whereas t-cinnamic acid, by comparison, affected the enzyme at high concentrations (K _I=1.8·10^-4).Abbreviations PAL phenylalanine ammonia lyase - L-AOPP L--aminooxy--phenylpropionic acid     

Neurotoxic Potential of Three Structural Analogs of β-N-oxalyl-α,β-Diaminopropanoic Acid (β-ODAP)

Lathyrism is a non-progressive motor neuron disease produced by consumption of the excitatory amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (-ODAP). To learn more about the mechanisms underlying Lathyrism three structural analogs of -ODAP were synthesized. Carboxymethyl-,-diaminopropanoic acid (CMDAP) evoked inward currents which were antagonized by APV (30 M), but not by CNQX (10 M). N-acetyl-,-diaminopropanoic acid (ADAP) evoked no detectable ionic currents but potentiated N-methyl-D-aspartate (NMDA)-activated currents. The potentiation of NMDA currents by ADAP was blocked by 7-chlorokynurenic acid. Carboxymethylcysteine (CMC) did not activate any detectable ionic currents. None of the three -ODAP analogs produced visible symptoms of toxicity in day old chicks when administered for 2–3 consecutive days. Ligand binding studies demonstrated that all the three compounds were effective to in displacing [³H]glutamate. The maximum inhibition was 92% for CMDAP, 61% for ADAP, 65% for CMC and 99% for -ODAP. These data indicate that analogs of -ODAP may interact with glutamate receptors without producing neurotoxicity.     

Maple syrup urine disease: Branched-chain amino acid concentrations and metabolism in cultured human lymphoblasts

The intracellular concentration of free leucine, isoleucine, and valine and their metabolism were studied in lymphoblast cultures established from peripheral blood of an individual with maple syrup urine disease (MSUD) and a control subject. Branched-chain -keto acid decarboxylase activity in the MSUD cells was 10% or less of the control value as measured by the ability of the cells to release ¹⁴CO₂ from the corresponding [1-¹⁴C]labeled branched-chain amino acid. The intracellular concentrations of free leucine and isoleucine were increased three-fold in MSUD lymphoblasts as compared to control cells. Free valine was present in only trace amounts of less than 0.1 mMin both cell lines. Exposure of normal and mutant cells to a 10 mMload of leucine, isoleucine, and valine resulted in a comparable concentration within cells after 24 hr. Concentrations returned to base values in normal cells 12 hr after removal of load, but leucine remained elevated in MSUD cells after 3 days. Leucine and its keto acid, -ketoisocaproic acid, added to the culture medium gave significant growth inhibition of MSUD lymphoblasts but not of normal cells, in the millimolar range. Isoleucine, valine, and their keto acids had no effect.This investigation was supported in part by Grants AM-13622, AM-05646, and GM-17702 from the United States Public Health Service, Veterans Administration Grant M.R.I.S. No. 3181 to Dr. Nathan Gochman, and grants from the National Foundation and the Kroc Foundation. S. D. S. is a Postdoctoral Research Fellow supported by United States Public Health Service Training Grant AM-05646.     

Effects of natural interferon α, natural tumor necrosis factor α and their combination on human mesothelioma xenografts in nude mice

Effects of human natural interferon (nIFN) alone, human natural tumor necrosis factor (nTNF) alone and their combination (OH-1) were tested on three human mesothelioma lines implanted in nude mice. Tumors were transplanted subcutaneously by trocar on treatment day –12. nIFN was given intraperitoneally (i.p.) at a dose of 2 × 10⁷ or 2 × 10⁸ IU kg^–1 day^–1, 5 days a week for 3 weeks. nTNF was given i.p. at a dose of 2 × 10⁷ or 2 × 10⁸ U kg^–1 day^–1 in the same schedule as that of nIFN. Tumor diameters were serially measured and tumor volumes were calculated. Antitumor effects were assessed by two methods: comparison of final tumor volumes in treated and control groups (T/C), and changes in median average total tumor volume. The treatment produced no clinically discernible toxicities. nIFN had strong inhibitory activity against all three human mesothelioma lines. nTNF alone had modest activity only at the high dose used. The combination of the two produced activity essentially similar to that produced by nIFN alone. High-dose nIFN may have a role as an active agent in the treatment of patients with mesothelioma.