Search

Background: β Amyloid (Aβ)-mediated neuronal hyperactivity, a key feature of the early stage of Alzheimer's disease (AD), is recently proposed to be initiated by the suppression of glutamate reuptake. Nevertheless, the underlying mechanism by which the impaired glutamate reuptake causes neuronal hyperactivity remains unclear. Chronic suppression of the glutamate reuptake causes accumulation of ambient glutamate that could diffuse from synaptic sites at the dendrites to the soma to elevate the tonic activation of somatic N-methyl-D-aspartate receptors (NMDARs). However, less attention has been paid to the potential role of tonic activity change in extrasynaptic glutamate receptors (GluRs) located at the neuronal soma on generation of neuronal hyperactivity., Methods: Whole-cell patch-clamp recordings were performed on CA1 pyramidal neurons in acute hippocampal slices exposed to TFB-threo-β-benzyloxyaspartic acid (TBOA) or human Aβ 1-42 peptide oligomer. A series of dendritic patch-clamp recordings were made at different distances from the soma to identify the location of the changes in synaptic inputs. Moreover, single-channel recording in the cell-attached mode was performed to investigate the activity changes of single NMDARs at the soma., Results: Blocking glutamate uptake with either TBOA or the human Aβ 1-42 peptide oligomer elicited potentiation of synaptic inputs in CA1 hippocampal neurons. Strikingly, this potentiation  specifically occurred at the soma, depending on the activation of somatic GluN2B-containing NMDARs (GluN2B-NMDARs) and accompanied by a substantial and persistent increment in the open probability of somatic NMDARs. Blocking the activity of GluN2B-NMDARs at the soma completely reversed both the TBOA-induced or the Aβ 1-42 -induced somatic potentiation and neuronal hyperactivity., Conclusions: The somatic potentiation of synaptic inputs may represent a novel amplification mechanism that elevates cell excitability and thus contributes to neuronal hyperactivity initiated by impaired glutamate reuptake in AD., (© 2021. The Author(s).)

Chemically cross-linked poly(aspartic acid) (PASP) gels were prepared by a solid-liquid phase separation technique, cryogelation, to achieve a supermacroporous interconnected pore structure. The precursor polymer of PASP, polysuccinimide (PSI) was cross-linked below the freezing point of the solvent and the forming crystals acted as templates for the pores. Dimethyl sulfoxide was chosen as solvent instead of the more commonly used water. Thus larger temperatures could be utilized for the preparation and the drawback of increase in specific volume of water upon freezing could be eliminated. The morphology of the hydrogels was characterized by scanning electron microscopy and interconnectivity of the pores was proven by the small flow resistance of the gels. Compression tests also confirmed the interconnected porous structure and the complete re-swelling and shape recovery of the supermacroporous PASP hydrogels. The prepared hydrogels are of interest for several biomedical applications as scaffolding materials because of their cytocompatibility, controllable morphology and pH-responsive character., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Peptide-nucleotide antibiotic microcin C (McC) is produced by some Escherichia coli strains. Inside a sensitive cell, McC is processed, releasing a nonhydrolyzable analog of aspartyl-adenylate, which inhibits aspartyl-tRNA synthetase. The product of mccE, a gene from the plasmid-borne McC biosynthetic cluster, acetylates processed McC, converting it into a nontoxic compound. MccE is homologous to chromosomally encoded acetyltransferases RimI, RimJ, and RimL, which acetylate, correspondingly, the N termini of ribosomal proteins S18, S5, and L12. Here, we show that E. coli RimL, but not other Rim acetyltransferases, provides a basal level of resistance to McC and various toxic nonhydrolyzable aminoacyl adenylates. RimL acts by acetylating processed McC, which along with ribosomal protein L12 should be considered a natural RimL substrate. When overproduced, RimL also makes cells resistant to albomycin, an antibiotic that upon intracellular processing gives rise to a seryl-thioribosyl pyrimidine that targets seryl-tRNA synthetase. We further show that E. coli YhhY, a protein related to Rim acetyltransferases but without a known function, is also able to detoxify several nonhydrolyzable aminoacyl adenylates but not processed McC. We propose that RimL and YhhY protect bacteria from various toxic aminoacyl nucleotides, either exogenous or those generated inside the cell during normal metabolism., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

N-acetyl-l-aspartic acid (NAA) is a component of the mammalian central nervous system (CNS) that has also been identified in a number of foods. This paper reports the outcome of a reproductive toxicology study conducted with NAA in Sprague-Dawley rats. NAA was added to diets at target doses of 100, 250 and 500 mg/kg of body weight/day and administered for two consecutive generations. A carrier control group was administered diet with no added NAA and a comparative control group was given aspartate (ASP), the constituent amino acid of NAA, at a target dose of 500 mg/kg of body weight/day. The study evaluated OECD 416 reproductive performance variables and additional segments to assess potential developmental effects, neurobehavioural and ophthalmologic function, and the concentrations of NAA or ASP in brain and plasma. No biologically significant differences were observed in any reproductive response variables, neurobehavioural tests, ophthalmologic examinations, body weights, feed consumption, or organ weights. Further, no test substance related mortalities or adverse clinical, neurohistopathologic or histopathologic findings were observed. Under the conditions of this study, the highest target dose of NAA, 500 mg/kg of body weight/day, represents the no-observed-adverse-effect-level (NOAEL) for reproductive and systemic toxicity, and neurotoxicity for Sprague-Dawley rats., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We investigated the systemic effects of subchronic dietary exposure to NAA in Sprague Dawley® rats. NAA was added to the diet at different concentrations to deliver target doses of 100, 250 and 500 mg/kg of body weight/day and was administered for 90 consecutive days. All rats (10/sex/group) survived until scheduled sacrifice. No diet-related differences in body weights, feed consumption and efficiency, clinical signs, or ophthalmologic findings were observed. No biologically significant differences or adverse effects were observed in functional observation battery (FOB) and motor activity evaluations, hematology, coagulation, clinical chemistry, urinalysis, organ weights, or gross pathology evaluations that were attributable to dietary exposure to NAA. Treatment-related increased incidence and degree of acinar cell hypertrophy in salivary glands was observed in both male and female rats in the high dose group. Because there was no evidence of injury or cytotoxicity to the salivary glands, this finding was not considered to be an adverse effect. Based on these results and the actual average doses consumed, the no-observed-adverse-effect-levels (NOAEL) for systemic toxicity from subchronic dietary exposure to NAA were 451.6 and 490.8 mg/kg of body weight/day for male and female Sprague Dawley® rats, respectively., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Previously we demonstrated the potential of D-aspartic acid (D-Asp), an acidic amino acid to induce oxidative response in prepubertal rat testis in vitro. In the present study, we determined the extent of oxidative stress in the testis of prepubertal rats that were administered D-Asp (100 and 500 mg/kg bw/d, i.p. 7 days). D-Asp treatment significantly elevated the levels of reactive oxygen species, malondialdehyde and hydroperoxide in cytosol and mitochondria of testis, which were accompanied by enhanced glutathione levels, elevated activities of glutathione-dependent enzymes and catalase suggesting a state of oxidative stress. Further, the activities of D-aspartate oxidase and 3beta-hydroxy steroid dehydrogenase were elevated in the testis. The testis mitochondria of D-Asp-treated rats showed altered citric acid and complex enzyme activities, reduction in membrane potential, increased permeability and intracellular Ca(2+) levels. Collectively, these findings suggest the potential of D-Asp to induce oxidative perturbations in the testis of prepubertal rats and this mechanism may in part be responsible for the observed physiological effects.

Analytical studies have reported that N-acetyl-L-aspartic acid (NAA) is present at low concentrations in many foods. The current studies were conducted to assess the mutagenicity of NAA using standard OECD guideline in vitro bacterial and in vivo mammalian mutagenicity studies. For comparison and control data, mutagenicity studies were also conducted with its constituent amino acid L-aspartate (ASP) because NAA is metabolized to ASP. The combination of an in vitro method for assessing point mutations in bacteria and an in vivo method to assess clastogenicity in an animal model provided adequate evidence for mutagenicity hazard assessment of NAA. No evidence of mutagenicity was observed in either test system with either NAA or ASP. The results from the current studies demonstrate that the presence of NAA in foods is not likely to represent a risk for mutagenicity.

A subchronic oral toxicity study of l-aspartic acid (l-Asp) was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.05%, 1.25%, 2.5% and 5.0% concentrations for 90 days. Serum biochemistry showed treatment-related decreases of blood urea nitrogen, creatinine and uric acid levels in both sexes. In addition, incidences of urinary ketone and protein were significantly increased in treated both sexes, while relative kidney weight was significantly increased in the 5.0% male rat, and regenerative renal tubules with tubular dilation were histopathologically observed in male rats of the 2.5% or greater groups. The observed renal injury was confirmed not to be due to accumulation of alpha2u-globulin. Acinar cell hypertrophy of salivary glands was histopathologically evident in male and female rats of the 2.5% or greater groups. The present results indicate that l-Asp causes toxic effects on kidneys and possibly salivary glands at high dose levels in male and female Fischer 344 rats. Such toxic effects were observed only in animals given 2.5% and/or higher doses of l-Asp. In conclusion, the no-observed-adverse-effect-level (NOAEL) for l-Asp is 1.25% (696.6 mg/kg body weight/day for males and 715.2 mg/kg body weight/day for females) under the present experimental conditions.

N-acetyl-l-aspartic acid (NAA) is a constituent of the mammalian central nervous system (CNS) that has been identified in a number of commonly consumed foods. The current study reports the outcome of acute and repeated dose oral toxicology studies conducted with NAA in Sprague-Dawley (SD) rats. No mortalities or evidence of adverse effects were observed in SD rats following acute oral administration of 2000mg/kg NAA. In a separate study, NAA was added to the diets of SD rats (n=10/sex group) at concentrations corresponding to daily doses of 10, 100, or 1000mg/kg/day for 14 consecutive days and 100, 500, and 1000mg/kg/day for another 14 days. All rats survived until scheduled sacrifice and no differences in body weights, feed consumption values, or clinical signs were observed in any of the treatment groups. No biologically significant differences were observed in functional observational battery (FOB), motor activity evaluations, ophthalmologic examinations, hematology, coagulation, clinical chemistry, or organ weights of any of the NAA treatment groups. Further, no test substance-related gross or microscopic changes were observed in NAA exposure groups. Based on these results, NAA was not considered acutely toxic following oral exposure to 2000mg/kg and the no-observed-adverse-effect-level (NOAEL) for systemic toxicity from repeated dose dietary exposure to NAA is 1000mg/kg/day.

Purpose: We previously reported that an increase in the N-acetyl-L-aspartate (NAA) level due to the lack of aspartoacylase gene was found in the brain of the tremor rat (tm/tm), which is a mutant with a causative gene named tm that shows epileptic seizures. Therefore, NAA is suggested to be one of the factors involved in the induction of epileptic seizures. Patch-clamp studies were performed to determine whether NAA produces an excitatory effect on acutely dissociated rat hippocampal neurons., Methods: Acutely dissociated hippocampal neurons were prepared from normal Wistar rats aged 3-4 weeks. NAA-induced currents were investigated by using the whole-cell voltage-clamp recording technique., Results: Application of NAA at concentrations of 100 nM to 1 mM through a U-tube for 2 s produced an inward current in a concentration-dependent manner at a holding potential of -60 mV. When the current-voltage relation was examined, the reversal potential of the NAA-induced current was found to be approximately 0 mV. The NAA-induced current was inhibited by bath application of the metabotropic glutamate receptor (mGluR) antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG) and by intracellular application of guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS), a nonhydrolyzable GDP analogue. However, the NAA-induced current remained unaffected by glutamic acid diethyl ester, a non-N-methyl-D-aspartate (NMDA)-subtype ionotropic glutamate receptor antagonist, or the voltage-dependent ion channel blockers tetrodotoxin, CdCl2, and tetraethylammonium-chloride. Conversely, the mGluR agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) also induced an inward current, with a reversal potential of 0 mV. The ACPD-induced current also was inhibited by MCPG., Conclusions: These results suggest that NAA acts on the G protein-coupled mGluRs to induce an inward current that results in excitation of the neurons, thereby contributing to the occurrence of epileptic seizures.

cis-Diamminedichloroplatinum (II) (cisplatin, CDDP), a potent anticancer agent, was bound to the aspartic acid residues of poly(ethylene glycol)-poly(aspartic acid) (PEG-P(ASP)) block copolymer by ligand substitution reaction at the platinum atom of CDDP. The polymeric drug thus obtained was observed to form a micelle structure in aqueous medium, showing excellent water solubility. In the present study, in vitro and in vivo antitumor activity against several human tumor cell lines, toxicity and pharmacokinetic characteristics in rodents of CDDP-incorporated polymeric micelles (CDDP / m) were evaluated in comparison with those of CDDP. In vitro, CDDP / m exhibited 10 - 17% of the cytotoxicity of CDDP against human tumor cell lines. CDDP / m given by intravenous (i.v.) injection yielded higher and more sustained serum levels than CDDP. In vivo CDDP / m treatment resulted in higher and more sustained levels in tumor tissue than CDDP, and showed similar antitumor activity to CDDP against MKN 45 human gastric cancer xenograft. CDDP / m treatment caused much less renal damage than CDDP. These results indicate that CDDP / m treatment can reduce CDDP-induced nephrotoxicity without compromising the anticancer cytotoxicity of CDDP.

In human fibroblasts, N:-phosphoacetyl-L-aspartate (PALA) and gamma-radiation induce reversible and irreversible p53-mediated G(1) cell cycle arrest, respectively. By coupling the premature chromosome condensation technique to fluorescence in situ hybridization, we found no evidence of DNA damage after PALA treatment. We used representational difference analysis (cDNA-RDA) to study changes in gene expression after PALA treatment and gamma-radiation in normal human fibroblasts. The mammary-derived growth inhibitor (MDGI) gene was expressed in PALA-treated cells. Ectopic MDGI expression arrested PALA-treated but not irradiated RKO cells. Expression of an antisense RNA against MDGI resulted in partial G(1) escape of PALA-treated human fibroblasts. The tumor necrosis factor stimulated gene 6, TSG-6, seems to be under the control of p53 and is only and specifically induced upon PALA treatment. In irradiated cells we have identified 'novel' genes that are differentially expressed, along with known genes not previously linked to cell cycle control. Some of these 'novel' genes correspond to clones in the expressed sequence tag (EST) database; one of them shows identity with ESTs mapping to a region on chromosome 7, where gene(s) involved in replicative senescence and frequently deleted in tumors are located. Thus, PALA treatment and gamma-irradiation elicit a pattern of differential gene expression that could contribute to a quiescence or senescence-like phenotype.

Background: Considerable evidence exists that neurotoxicity of excitatory amino acids is related to the neuronal injury, including paraplegia. However, little is known about aspartate neurotoxicity in the spinal cord in vivo. We evaluated the detrimental effects of exogenous aspartate on spinal cord neurons under metabolic stress., Methods: New Zealand white rabbits underwent an infrarenal aortic isolation. Group A animals (n = 7) received segmental aspartate 50 mmol/L) infusion for 10 minutes. Group B animals (n = 7) received saline as a negative control. Group C animals (n = 5) received segmental aspartate 100 mmol/L) infusion for 5 minutes. Group D animals (n = 7) were pretreated with segmental infusion of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptan-5,10-imine (MK-801) (6 mg/kg), a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist for 1 minute, followed by segmental infusion of aspartate (50 mmol/L) for 9 minutes. Group E animals (n = 7) received vehicle only, followed by aspartate (50 mmol/L) infusion as a control of group D. Neurologic status was assessed at 12, 24, and 48 hours after operation using the Tarlov score., Results: Group A animals exhibited paraplegia or paraparesis with marked neuronal necrosis. Group B and C animals recovered fully. Group D animals showed significantly better neurologic function (p = 0.0007) compared with group E animals that exhibited paraplegia or paraparesis., Conclusions: Exogenous aspartate can have detrimental effects on spinal cord neurons under metabolic stress. This model may be useful in assaying neuronal injury mediated by NMDA receptor in vivo.

Abnormalities in the p53 tumor suppressor gene have been shown to affect cellular processes related to cell cycle control and gene amplification. In this study we compare the status and function of wild-type p53 in MCF-7 breast cancer cells with sublines selected for resistance to chemotherapeutic agents having different mechanisms of action. Sublines that were resistant to melphalan, pyrazafurin, mitoxantrone, etoposide and PALA all retained expression of wild-type p53. Methotrexate-resistant MCF-7 cells were unusual heterozygotes that expressed a wild-type and dominant, in-frame p53 deletion mutant and the doxorubicin-resistant cells expressed only mutant p53. Analysis of the G1 checkpoint after treatment with ionizing radiation revealed that the pyrazafurin-, melphalan- and mitoxantrone-resistant cells arrested strongly in G1. The etoposide- and PALA-resistant cells had an intermediate G1 arrest phenotype and the methotrexate- and doxorubicin-resistant cells had a minimal G1 arrest phenotype. mRNA and protein analyses of downstream effector genes, including P21CIP1/Waf1, mdm2, Gadd 45 and the retinoblastoma protein, did not entirely differentiate sublines having a strong versus intermediate G1 arrest phenotype. Neither the p53 status nor the strength of the G1 arrest could be correlated with cell survival after ionizing radiation. When drug-sensitive MCF-7 cells were treated with the same chemotherapeutic agents, p53 and p21CIP1/Waf1 levels increased between 2- and 14-fold. Together these data suggest that other cellular factors likely play a role in overcoming the inhibitory effects of ionizing radiation on p53 in drug-resistant breast cancer cells.

Chemotherapeutic treatment of tumor cells leads either to tumor cell death (usually by apoptosis) or to the formation of drug-resistant subpopulations. Known mechanisms of cancer cell drug resistance include gene amplification and increased expression of drug transporters. On the other hand, normal cells survive many forms of chemotherapy with minimal damage probably because of their capacity for growth arrest and stringent control of apoptosis. Microcell hybrids between B78 (murine melanoma) and HSF5 (normal human fibroblasts) were analyzed to identify a new human chromosomal region involved in the promotion of drug-induced growth arrest and suppression of apoptosis. In these hybrids, the presence of human chromosome 3 was strongly associated with suppression of apoptosis via G1 and G2 growth arrest during exposure to the antimetabolite N-phosphonoacetyl-L-aspartate (PALA), suggesting that a gene(s) on chromosome 3 serves an antiproliferative role in a drug-responsive growth arrest pathway.

Cytosine deaminase (EC 3.5.4.1), a non-mammalian enzyme, catalyzes the deamination of cytosine and 5-fluorocytosine to form uracil and 5-fluorouracil, respectively. Eukaryotic cells have been genetically modified with a bacterial cytosine deaminase gene to express a functional enzyme. When the genetically modified cells are combined with 5-fluorocytosine, it creates a potent negative selection system, which may have important applications in cancer gene therapy. In this paper, we introduce a novel positive selection method based upon the expression of the cytosine deaminase gene. This method utilizes inhibitors in the pyrimidine de novo synthesis pathway to create a condition in which cells are dependent on the conversion of pyrimidine supplements to uracil by cytosine deaminase. Thus, only cells expressing the cytosine deaminase gene can be rescued in a positive selection medium.

Feeding bees with organic cupric salts provides long-term control of the parasite Varroa jacobsoni. A set of new algebraic parameters (M. Bounias C.R. Acad. Sci. 310(3), 65-70, 1990) completely describing the population lethality function has been calculated following chronic administration of cupric gluconate, aspartate, and isoleucinate, with or without dietary pollen. Mortality curves allowed the calculation of LT50 (time for 50% lethality) as well as Hill coefficients (h) of the curves and the LD50 as a function of time. The tangent at the inflexion point of the sigmoidal time/mortality curves (delta i) gave the maximum mortality acceleration as an additional parameter. No toxicity (i.e., no decrease of TL50 vs doses and no LD50 values) was found for cupric gluconate and isoleucinate with pollen, whereas increases in LT50 and decreases in delta indicated hormesis effects. Doses decreasing by half-time LT50, h, or delta were used as objective lethality indexes for comparisons of toxicity in the other cases. Routine acute toxicity at high dosage was also compared with phosalone and lindane effects 24 hr after treatment.

Transforming growth factor beta 1 (TGF-beta 1) regulates a multitude of diverse biological functions in mammalian cells, and there is good evidence that aberrant expression of this growth factor can play an important role in mechanisms of malignant progression. We show that a TGF-beta 1-overexpressing mouse 10T1/2 cell line transfected with a TGF-beta 1 sequence that allows the synthesis of bioactive growth factor exhibits reduced sensitivity to the cytotoxic effects of the drug N-(phosphonacetyl)-L-aspartate (PALA) in colony-forming experiments. Furthermore, six independent 10T1/2 TGF-beta 1-transfected cell lines containing TGF-beta 1 gene expression under the control of a zinc sulfate-responsive metallothionein promoter were selected. In all cases, sensitivity to PALA cytotoxic effects was significantly reduced when cells were cultured under conditions that led to elevated levels of TGF-beta 1 gene expression when compared to cells containing basal levels of this growth factor. Fluctuation analysis to determine the rate of PALA resistance was performed with several TGF-beta 1-transfected cell lines in which growth factor expression was regulated by the metallothionein promoter. We observed significantly higher rates of PALA resistance/cell/generation in cell populations expressing high levels of TGF-beta 1 than in the same cells expressing relatively low levels of this growth factor. The only mechanism known for PALA resistance in mouse cells involves the amplification of the gene coding for the protein target of PALA, CAD, a multifunctional polypeptide containing carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase. Southern blot analysis of colonies that survived normally cytotoxic concentrations of PALA exhibited CAD gene amplification. In total, these observations indicate that aberrant expression of TGF-beta 1 gene expression decreases the genetic stability of 10T1/2 cells, leading to increased rates of drug resistance and elevated gene amplification potential. The results of this study indicate a new malignancy related function for TGF-beta 1 alterations and suggest a novel role for aberrant expression of this growth factor in mechanisms of drug resistance and tumor progression.

Preclinical studies suggest that the biochemical effects of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate carbamoyltransferase (ACTase), may increase the metabolic activation of 5-fluorouracil (5-FU) and enhance its cytotoxicity through both RNA- and DNA-directed mechanisms. In this Phase I trial, 22 evaluable patients with adenocarcinoma of the gastrointestinal tract were entered at escalating doses of 5-FU starting at 1150 mg/m2/day given as a concurrent 72-h i.v. infusion with a fixed dose of leucovorin (LCV), 500 mg/m2/day. The dose of 5-FU was escalated within patients according to individual tolerance, and then PALA at 250 mg/m2 was added 24 h prior to the initiation of the 5-FU/LCV infusion of the subsequent cycle. Dose-limiting mucositis and myelosuppression occurred during the initial cycle in 3 of 5 patients treated with 2300 mg/m2/day 5-FU; therefore, the recommended dose of 5-FU with concurrent LCV is 2000 mg/m2/day. Twenty-seven additional patients were then treated with escalating doses of PALA ranging from 375 to 2848 mg/m2, i.v., followed 24 h later by 2000 mg/m2/day 5-FU with high-dose LCV. Dose-limiting mucositis and myelosuppression occurred during the initial cycle in 2 of 3 patients entered at 2848 mg/m2 PALA. Dose-limiting mucositis and skin rash ultimately required both PALA and 5-FU dose reductions in 4 of 6 patients treated with 1899 mg/m2 PALA. Toxicity was similar, however, in patients receiving PALA at doses ranging from 375 to 1266 mg/m2. The mean steady-state plasma concentration of 5-FU at 2000 mg/m2/day was 6.5 +/- 0.9 microM; patients with 5-FU levels > 9 microM had a significantly higher incidence of serious gastrointestinal and hematological toxicity. Compared to each patient's own baseline, a significant trend for decreasing ACTase activity with increasing PALA dose was evident using cytosol isolated from peripheral blood mononuclear cells 24 h after PALA treatment (P2 = 0.01). PALA < or = 844 mg/m2 failed to appreciably inhibit ACTase activity at 24 h in most patients; furthermore, a decrease in ACTase activity by > 50% from baseline was seen in only 29% of cycles. More consistent inhibition of ACTase activity was seen with PALA > or = 1266 mg/m2. Even with the highest PALA doses, however, ACTase activity returned to baseline by 96 h in most patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Normal and SV40-infected human fibroblasts were grown in the presence of the drug N-(phosphonoacetyl)-L-aspartate (PALA) and examined for evidence of genetic instability. Both cell populations were precrisis and showed a normal, diploid karyotype at early passage. In contrast to the normal IMR-90 cells, which showed growth arrest and did not form colonies in PALA, the SV40-infected IMR-90 cells formed colonies at a very high frequency and continued to cycle in the drug. The drug-resistant colonies senesced after continued growth in culture, indicating that this change in ability to amplify preceded immortalization. This is the first observation of mortal human cells overcoming the drug-induced growth arrest. Although all previously isolated PALA-resistant colonies demonstrated CAD gene amplification as the mechanism of the drug-resistant phenotype, these SV40-infected human cells also showed alternative mechanisms, including increases in gene copy number by aneuploidy and formation of an isochromosome 2p.

Aspergillomarasmine-A and -B (AM-A and -B), which were isolated from the cultured broth of an unidentified fungus N877, showed apparent inhibition against endothelin-converting enzyme (ECE) from bovine endothelial cells as measured by the formation of endothelin-1 (ET-1) converted from big endothelin-1 (bET-1), with IC50 values of 3.4 and 2.5 microM for AM-A and -B, respectively. EDTA also inhibited ECE (IC50 = 1.1 microM), but the inhibitions by AM-A, AM-B and EDTA were each abolished by the addition of 10 microM Zn2+ to the reaction mixture. In mice, AM-A and -B dose-dependently (10-50 mg/kg, i.v.) caused significant prolongation of the latency to sudden death induced by i.v. bET-1 (25 nmol/kg), but not that by ET-1 (5 nmol/kg), accompanied by a decrease in plasma immunoreactive ET-1 formation, while EDTA (24 mg/kg) failed to do so. In mice, the LD50 value of AM-A was calculated to be 159.8 mg/kg, i.v., which was much larger than that of EDTA (28.5 mg/kg, i.v.), indicating the low toxicity of AM-A. AM-A (30 mg/kg, i.v.) also suppressed bET-1-induced hemoconcentration and hypertension in mice and rats, respectively. These findings suggest that although ECE inhibition by AM-A was mainly attributable to its chelating activity, it showed apparent in vivo activities due to ECE inhibition with low toxicity.

Human embryo fibroblasts of common genetic origin but exhibiting a range of phenotypes from normal to aggressively tumorigenic have been used to study resistance to the cytotoxic drugs methotrexate and N-(phosphonacetyl)-L-aspartate. Measurement of the intrinsic sensitivities of these cells to the two drugs in standard survival assays, in normal fetal bovine serum, showed increasing resistance to parallel increasing tumor-igenicity. Tumor cells were totally resistant to 10 mM N-(phosphonacetyl)-L-aspartate whereas the 50% lethal dose for methotrexate for the tumor cells was 500 nM compared with 50 nM for the normal diploid parent cell line. The difference in resistance between the immortal and tumorigenic cell lines was eliminated for both methotrexate and N-(phosphonacetyl)-L-aspartate, when the experiments were repeated in the presence of dialyzed fetal bovine serum, but could be restored by the addition of either hypoxanthine (100 microM) or uridine (10 microM). This suggested an important role for the salvage pathways of purine and pyrimidine biosynthesis in the increased resistance of the more tumorigenic cell lines. The implications of these data in relation to cancer chemotherapy will be discussed.

Two separate experiments were conducted to determine the ability of magnesium aspartate hydrochloride (Mg-Asp-HCl) to antagonize the effects of thermal stress in laying hens. In both experiments, hens were exposed to either a cyclic ambient temperature of 27 C to 35 C (hyperthermic group) or 23 C (control group) for 7 days. In Experiment 1, hens were given a single injection of either saline, 40 mg, or 80 mg Mg-Asp-HCl. Experiment 2 hens received a single injection of saline, 40 mg Mg-Asp-HCl, or twice daily injection of 40 mg Mg-Asp-HCl. In both experiments, heat-stressed hens had elevated body temperature and reduced body weight, feed consumption, and circulating mean Mg concentrations. Hyperthermic hens also had reduced egg weights in Experiment 1. A single daily injection of 40 mg of Mg-Asp-HCl significantly reduced body weight loss in heat stressed hens by 41 and 51%, in Experiment 1 and 2, respectively, when compared with heat-stressed hens not receiving Mg-Asp-HCl (controls). The Mg-Asp-HCl treatment did not affect overall feed intake, egg production, or body temperature in either experiment. The highest dose of Mg-Asp-HCl (80 mg) elevated circulating magnesium concentrations. A single daily injection of 80 mg of Mg-Asp-HCl or twice daily injections of 40 mg Mg-Asp-HCl did not provide any additional benefit when compared with a single daily dose of 40 mg. The antistress effects of Mg-Asp-HCl are apparent in these experiments as evidenced by the increased body weight of heat-stressed hens. Use of this compound may prove to be beneficial in maintaining the performance characteristics of poultry exposed to elevated ambient temperature.

High concentrations of potent N-methyl-D-aspartate (NMDA) agonists can trigger degeneration of cultured mouse cortical neurons after an exposure of only a few minutes; in contrast, selective non-NMDA agonists or low levels of NMDA agonists require exposures of several hours to induce comparable damage. The dihydropyridine calcium channel antagonist nifedipine was used to test whether this slow neurotoxicity is mediated by a calcium influx through voltage-gated channels. Nifedipine had little effect on the widespread neuronal degeneration induced by brief exposure to high concentrations of NMDA but substantially attenuated the neurotoxicity produced by 24-hour exposure to submaximal concentrations of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate, kainate, or quinolinate. Calcium ion influx through dihydropyridine-sensitive, voltage-dependent calcium channels may be an important step in the neuronal injury induced by the prolonged activation of NMDA or non-NMDA glutamate receptors.

Eight-day-old mice were given by gavage glutamate and aspartate mixtures providing each amino acid at 125, 250 or 500 mg/kg body weight (250, 500 and 1000 mg total dicarboxylic amino acids/kg) and the degree and extent of neuronal necrosis were determined. Similar studies were carried out in mice given monosodium L-glutamate at 250 or 500 mg/kg body weight. Plasma aspartate and glutamate concentrations were determined at each dose level. No animal given either glutamate or the glutamate plus aspartate mixture at 250 mg/kg developed neuronal necrosis. However, neuronal necrosis developed in 30% of animals given glutamate at 500 mg/kg (12+/-2 necrotic neurons/section in the region of maximal damage) and in 17% of animals given 250 mg glutamate/kg plus 250 mg aspartate/kg (11-13 necrotic neurons/section in the region of maximal damage). The threshold mean peak plasma glutamate plus aspartate concentration associated with neuronal necrosis was 128+/-24 mumol/dl. Using these data, and previously published data for aspartate-induced neurotoxicity (Finkelstein et al. Toxicology 1983, 29, 109), the individual threshold plasma glutamate and aspartate concentrations associated with neuronal necrosis were calculated to be 110 mumol/dl for aspartate and 75 mumol/dl for glutamate.

It has been suggested on the basis of in vitro studies that tricyclic antidepressants interact with the N-methyl-D-aspartic acid (NMDA)-receptor complex to block the action of NMDA. The present study showed that tricyclic antidepressants prevented lethality produced by a large dose of NMDA. The potency of the drugs in preventing NMDA-induced lethality correlated with the inhibition of [3H]-MK-801 binding at the NMDA receptor complex, and not with effects on amine uptake. These in vivo data support the in vitro data of Reynolds & Miller (1988a,b).

Rats of various ages were treated orally or intraperitoneally with potassium aspartate. The dose required to induce hypothalamic lesion varied considerably by the age of animals and route of administration. Additional experiment, in which the animals were orally treated three times a day with potassium aspartate in dose levels between the maximum safety dose and minimum lesion-producing dose in the preceding single dose study, revealed no hypothalamic lesion at all in any animals of each age group. In this condition, the maximum safety dose was 3--5 times as large as that in single dosage administration experiment. Regarding the safety evaluation of potassium aspartate preparations, brief discussions on some points in extrapolation of the results of the present experimental study to the clinical use were made.

A new type of drug acid-labile arm-carrier is described. A hydrophilic polymer [polytrisacryl, or poly(acryloyl 2-amido-2-hydroxymethyl-1,3-propanediol)], used as a model, is substituted with p-nitrobenzyl groups. The nitrobenzyl groups are reduced to aminobenzyl and transformed into benzylisothiocyanate groups which are allowed to react with aminoacyl daunorubicin. The excess of benzylisothiocyanate is transformed into benzylthiocarbamoyl N-methyl glucamine. A conjugate containing benzylthiocarbamoyl-aspartyl daunorubicin is stable at neutral pH, and releases free daunorubicin when it is exposed to pH 5 or below. This conjugate is about 200-fold less toxic than free daunorubicin, on a molecular basis, when it is added to the medium of Lewis lung carcinoma (3LL) cells in culture.

A Phase I clinical trial of N-(phosphonacetyl)-L-aspartate, an antimetabolite which inhibits a key enzyme in the de novo pathway of pyrimidine biosynthesis, was conducted. N-(Phosphonacetyl)-L-aspartate was given as an i.v. 15-min infusion once daily for five days; cycles of treatment were repeated every three weeks. Thirty-four patients received treatment. Dose-limiting toxicity was observed at 1500 to 2000 mg/sq m/day and was manifested by skin rash, diarrhea, and stomatitis. Rash and diarrhea usually began during the first week of treatment and persisted up to Day 17 of a cycle of therapy. No consistent hematopoietic, hepatic, or renal toxicity was observed. One partial response in a patient with colon carcinoma was seen and continues at more than eight months. Stable disease was observed in three patients with colon carcinoma, two patients with hypernephroma, one patient with pancreatic carcinoma, and one patient with melanoma. The predictability and reversibility of toxicity and the suggestion of antitumor activity in humans are observations which support the further evaluation of N-(phosphonacetyl)-L-aspartate in Phase II studies.

The effect of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and its nontumor promoting derivative 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate on the frequency of mouse and hamster cells resistant to methotrexate (MTX), N-(phosphonacetyl)-L-aspartate, and cadmium has been examined. TPA alone at concentrations up to 1.0 microgram/ml had no significant effect on the plating efficiency of either mouse or hamster cells. Exposure of 3T3 and 3T6 mouse and V79 and Chinese hamster ovary cells at low density to the 3 compounds in the presence of TPA (0.1 microgram/ml) did not result in any increase in the recovery of resistant colonies. When plated at high density, exposure to drug selection in the presence of TPA resulted in a 3- to 10-fold increase overall in the incidence of MTX-, N-(phosphonacetyl)-L-aspartate-, and cadmium-resistant mouse cells. However, an increase greater than 3-fold was not observed in hamster cells exposed to drug plus TPA under the same conditions. 4-O-Methyl-12-O-tetradecanoylphorbol-13-acetate had no significant effect on the frequency of MTX-resistant cells. Seventy V79 cell clones surviving MTX (200 to 400 nM) alone and 79 surviving MTX plus TPA were isolated and retested for resistance to MTX. None were stable. In contrast, 6 out of 42 mouse colonies isolated from MTX alone and 55 out of 99 isolated from MTX plus TPA showed stable resistance on retesting in MTX. The implications of these results in relation to possible mechanisms of tumor promotion are discussed.