Comparative effect of L-CCG-I, DCG-IV and gamma-carboxy-L-glutamate on all cloned metabotropic glutamate receptor subtypes

We examined the different personality dimensions between depression and anxiety with Cloninger's seven-factor model of temperament and character. The Temperament and Character Inventory (TCI), which measures four temperament and three character dimensions of Cloninger's personality theory (125-item short version), the Self-rating Depression Scale (SDS), and the State-Trait Anxiety Inventory (STAI) were administered to 223 Japanese students. With hierarchical regression analysis, the SDS score was predicted by scores of Harm-Avoidance, Self-Directedness, and Self-Transcendence, even after controlling for the STAI score. The STAI score was predicted by scores of Self-Directedness and Cooperativeness, even after controlling for the SDS score. More importance should be attached to these dimensions of character because they might contribute to both depression and anxiety.     

Behavioral activity of 1S,3R-ACPD, an agonist of metabotropic glutamate receptors

The influence of intracerebroventricular (i.c.v.) injections of 1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), an agonist of metabotropic glutamate receptors, on the activity of the central nervous system was examined in rats. 1S,3R-ACPD at the i.c.v. doses of 100 and 200 nmole significantly decreased apomorphine-induced stereotypy, and at a dose 200 nmole significantly diminished catalepsy caused by haloperidol, two behavioral symptoms controlled mainly by central dopaminergic system. The tested compound had no influence on the locomotor and exploratory activity of rats estimated in open field. 1S,3R-ACPD significantly improved acquisition at the i.c.v. dose of 100 nmole, and consolidation of information at the i.c.v. doses of 100 and 200 nmole. The tested compound had no influence on retention processes in passive avoidance situation. This compound also did not improve recognition memory tested in the object recognition test. These results indicated that 1S,3R-ACPD improved memory motivated affectively but had no influence on recognition memory and the diminished dopaminergic transmission.     

2,3'-disubstituted-2-(2'-carboxycyclopropyl)glycines as potent and selective antagonists of metabotropic glutamate receptors

2-(9-Xanthylmethyl)-2-(2'-carboxycyclopropyl) glycine 6e is a novel metabotropic glutamate receptor antagonist. A series of alpha, C-3' disubstituted (carboxycyclopropyl)glycines 6f-n were prepared. Antagonist activity was observed for all these compounds at group 2 and group 3 mGluRs. Although they were slightly less active on group 2 mGluRs than non C-3' substituted 6e, the compounds 6f-n were more selective with lesser or no activity on group 1 mGluR subtypes (IC50 values greater than 100 microns).     

LY354740, a group II metabotropic glutamate receptor agonist with potential antiparkinsonian properties in rats

The aim of this study was to examine whether (+)-2-aminobicyclo[3.1.0]-hexane-2,6-dicarboxylate monohydrate (LY354740), a selective agonist of group II metabotropic glutamate receptors, possesses antiparkinsonian properties. Parkinsonian-like muscle rigidity was induced by pretreatment with haloperidol (1 mg/kg i.p.). It was measured as increased resistance developed by the rat's hind leg to passive extension and flexion. LY354740 (5 and 10 mg/kg i.p.) dose-dependently diminished the haloperidol-induced muscle rigidity. The present results suggest that LY354740 counteracts the muscle rigidity in an animal model of parkinsonism.     

Anxiolytic and side-effect profile of LY354740: a potent, highly selective, orally active agonist for group II metabotropic glutamate receptors

LY354740 is a conformationally constrained analog of glutamate which is a potent agonist for group II cAMP coupled metabotropic glutamate receptors (mGluRs). The discovery of this novel pharmacological agent has allowed the exploration of the functional consequences of activating group II mGluRs in vivo. In an effort to evaluate the clinical utility of LY354740 as an anxiolytic, we examined its effects in the fear potentiated startle and elevated plus maze models of anxiety and compared the results with the clinically prescribed anxiolytic diazepam. In the fear potentiated startle and elevated plus maze models, both LY354740 and diazepam produced significant anxiolytic activity (ED50 values of 0.3 and 0.4 mg/kg p. o. for fear potentiated startle and 0.2 and 0.5 mg/kg for the elevated plus maze, respectively). The duration of pharmacological effect for LY354740 in the efficacy models was 4 to 8 hr. In contrast to diazepam, acute administration of LY354740 did not produce sedation, cause deficits in neuromuscular coordination, interact with central nervous system depressants, produce memory impairment or change convulsive thresholds at doses 100- to 1000-fold the efficacious doses in animal models of anxiety. Thus, LY354740 has anxiolytic activity in animal models that are sensitive to benzodiazepines such as diazepam. However, at anxiolytic doses in these models, LY354740 produced none of the unwanted secondary pharmacology associated with diazepam. These data indicate a functional role for group II mGluRs in fear/anxiety responses in animals and suggest that compounds in this class may be beneficial in the treatment of anxiety-related disorders in humans without the side effects seen with currently prescribed medications.     

3H]LY341495, a highly potent, selective and novel radioligand for labeling Group II metabotropic glutamate receptors

We report herein the synthesis and pharmacological characterization of a tritiated version of the potent and selective cyclopropyl amino acid LY341495 as a radioligand to label group II metabotropic glutamate receptors in rat brain homogenates.     

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-dicarboxycyclopropyl)glycine binding in rat brain

[(2S,2'R,3'R)-2-(2',3'-[3H]Dicarboxycyclopropyl)glycine ([3H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K(D) value of 180 +/- 33 nM and a Bmax of 780 +/- 70 fmol/mg of protein. The nonspecific binding, measured using 100 microM LY354740, was <30%. NMDA, AMPA, kainate, L(-)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [3H]DCG IV binding up to 1 mM. However, several compounds inhibited [3H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S,2'S)-2-methyl-2-(2-carboxycyclopropyl)glycine > L-glutamate = ibotenate > quisqualate > (RS)-alpha-methyl-4-phosphonophenylglycine = L(+)-2-amino-3-phosphonopropionic acid > (S)-alpha-methyl-4-carboxyphenylglycine > (2S)-alpha-ethylglutamic acid > L(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-L-aspartyl-L-glutamic acid inhibited the binding in a biphasic manner with an IC50 of 0.2 microM for the high-affinity component. The binding was also affected by GTPgammaS, reducing agents, and CdCl2. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPgammaS show that [3H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.     

Antagonist properties of a phosphono isoxazole amino acid at glutamate R1-4 (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid receptor subtypes

All three subtypes of beta-adrenoceptors are coupled to stimulation of adenylyl cyclase activity via the stimulatory guanine-nucleotide-binding protein. Nevertheless, the beta3 adrenoceptor (beta3-AR) differs significantly from the other subtypes in terms of pharmacology. Most strikingly, it recognizes as agonists several compounds acting as potent beta1-AR and beta2-AR antagonists. Furthermore, the human beta3-AR is quite different from the animal beta3-AR. Molecular modelling studies followed by site-directed mutagenesis was used here to identify some of the amino acid residues which may be implicated in ligand binding and signal transduction of the beta3-AR. Three contiguous residues, valine-leucine-alanine, which are present in the first transmembrane domain at positions 48-50 of the human receptor but are absent in all known rodent sequences, were thought to be important for species specificity. When these three residues were deleted from the human receptor, no 'rodent-like' pharmacological profile was obtained in terms of either binding or adenylyl cyclase activation. Glycine at position 53, also in the first transmembrane domain in the human beta3-AR, has been suggested to participate in beta2-/beta3-AR subtype selectivity. Replacement of this glycine residue by phenylalanine, which is the residue present at the homologous position in the human beta2-AR, left the beta3-AR pharmacological profile unaltered in terms of specificity and selectivity. Aspartate residue 117, in the third transmembrane domain, has been found to be essential for ligand binding and consequently adenylyl cyclase activation in several bioamine receptors. When this residue was replaced by a leucine residue in the beta3-AR, ligand binding and signal transduction were suppressed. Finally, replacement of asparagine at position 312 in the sixth transmembrane domain by an alanine residue, led to alterations in the signal-transduction pathway.     

Synthesis of the four isomers of 4-aminopyrrolidine-2,4-dicarboxylate: identification of a potent, highly selective, and systemically-active agonist for metabotropic glutamate receptors negatively coupled to adenylate cyclase

The four isomers of 4-aminopyrrolidine-2,4-dicarboxylate (APDC) were prepared and evaluated for their effects at glutamate receptors in vitro. (2R,4R)-APDC (2a), an aza analog of the nonselective mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (1S,3R)-ACPD, 1), was found to possess relatively high affinity for metabotropic glutamate receptors (mGluRs) (ACPD-sensitive [3H]glutamate binding IC50 = 6.49 +/- 1.21 microM) with no effects on radioligand binding to NMDA, AMPA, or kainate receptors up to 100 microM. None of the other APDC isomers showed significant mGluR binding affinity, indicating that this interaction is highly stereospecific. Both 1 and 2a were effective in decreasing forskolin-stimulated cAMP formation in the adult rat cerebral cortex (EC50 = 8.17 +/- 2.21 microM for 1; EC50 = 14.51 +/- 5.54 microM for 2a); however, while 1 was also effective in stimulating basal tritiated inositol monophosphate production in the neonatal rat cerebral cortex (EC50 = 27.7 +/- 5.2 microM), 2a (up to 100 microM) was ineffective in stimulating phosphoinositide hydrolysis in this tissue preparation, further supporting our previous observations that 2a is a highly selective agonist for mGluRs negatively coupled to adenylate cyclase. Microelectrophoretic application of either 1 or 2a to intact rat spinal neurons produced an augmentation of AMPA-induced excitation (95 +/- 10% increase for 1, 52 +/- 6% increase for 2a). Intracerebral injection of 1 (400 nmol) produced characteristic limbic seizures in mice which are not mimicked by 2a (200-1600 nmol, ic). However, the limbic seizures induced by 1 were blocked by systemically administered 2a in a dose-dependent manner (EC50 = 271 mg/kg, ip). It is concluded that (2R,4R)-APDC (2a) is a highly selective, systemically-active agonist of mGluRs negatively coupled to adenylate cyclase and that selective activation of these receptors in vivo can result in anticonvulsant effects.     

Glutamate metabotropic receptor agonist 1S,3R-ACPD induces internucleosomal DNA fragmentation and cell death in rat striatum

Glutamate metabotropic receptor mediated mechanisms have been implicated in both neuroprotection and neurotoxicity. To characterize these mechanisms further in vivo, the effects of an intrastriatally injected metabotropic receptor agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (1S,3R-ACPD), were studied alone and together with N-methyl-D-aspartate (NMDA) or kainic acid (KA) receptor agonists on DNA fragmentation and nerve cell death. 1S,3R-ACPD induced internucleosomal DNA fragmentation of striatal cells in a dose-dependent manner. TUNEL and propidium iodide staining showed DNA fragmentation and profound nuclear condensation around the injection site. Fragmented nuclei were occasionally seen under light microscopy. Internucleosomal DNA fragmentation induced by 1S,3R-ACPD was attenuated by the protein synthesis inhibitor cycloheximide as well as by the non-selective and selective metabotropic receptor antagonists L-(+)-2-amino-3-phosphonopionic acid (L-AP3), (RS)-aminoindan-1,5-dicarboxylic acid and (RS)-alpha-methylserine-o-phosphate monophenyl ester, respectively. The 1S,3R-ACPD (100-900 nmol) induced death of striatal neurons was suggested by the reduction in NMDA and D1 dopamine receptors by up to 13% (P < 0.05) and 20% (P < 0.05) as well as by the decline in GAD67 mRNA (25%, P < 0.01) and proenkephalin mRNA levels (35%, P < 0.01). Interestingly, 1S,3R-ACPD attenuated internucleosomal DNA fragmentation induced by NMDA, but potentiated that induced by KA. These results suggest that metabotropic receptor stimulation leads to the death of striatal neurons by a mechanism having the biochemical stigmata of apoptosis. Moreover, metabotropic receptor stimulation evidently exerts opposite effects on pre- or postsynaptic mechanisms contributing to the NMDA and KA-induced apoptotic-like death of these neurons.     

Differential effects of learning and recall of a spatial task on phosphoinositide hydrolysis induced by the metabotropic glutamate receptor agonist 1S,3R-ACPD (1S,3R-1-amino-cyclopentane-1,3-discarboxylic acid) in the hippocampus and the prefrontal cortex of rats

Phosphoinositide (PI) hydrolysis, stimulated by 1S,3R-1-amino-cyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), an agonist of metabotropic glutamate receptors (mGluRs), was measured in hippocampal and prefrontal cortical slices obtained from rats which had been trained for 8 days in a Morris water maze and had learned an allocentric spatial task. Brain slices were pre-labeled with myo-3H-inositol and 1S,3R-ACPD (100 microM) stimulation was assessed by measuring the accumulation of [3H]inositol phosphates ([3H]IPs) in the presence of Li+. Measurements conducted 24 h following the last training session revealed no differences in 1S,3R-ACPD-stimulated formation of [3H]IPs, either in the hippocampus or in the prefrontal cortex. However, a diminished response to mGluRs stimulation was detected in the hippocampus of animals re-trained after an 11-day interval. The decrease was not evident in the prefrontal cortex. These data indicate a differential involvement of the hippocampus and the prefrontal cortex in the processing of spatial information and correspond to the functional differences attributed to these areas.     

Converting parathyroid hormone-related peptide (PTHrP) into a potent PTH-2 receptor agonist

Most of the bone and kidney-related functions of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) are thought to be mediated by the PTH/PTHrP receptor. Recently, a homologous receptor, the PTH-2 receptor, was obtained from rat and human brain cDNA libraries. This receptor displayed the remarkable property of responding potently to PTH, but not to PTHrP. To begin to define residues involved in the ligand specificity of the PTH-2 receptor, we studied the interaction of several PTH/PTHrP hybrid ligands and other related peptide analogs with the human PTH-2 receptor. The results showed that two sites in PTH and PTHrP fully account for the different potencies that the two ligands exhibited with PTH-2 receptors; residue 5 (His in PTHrP and Ile in PTH) determined signaling capability, while residue 23 (Phe in PTHrP and Trp in PTH) determined binding affinity. By changing these two residues of PTHrP to the corresponding residues of PTH, we were able to convert PTHrP into a ligand that avidly bound to the PTH-2 receptor and fully and potently stimulated cAMP formation. Changing residue 23 alone yielded [Trp23]hPTHrP-(1-36), which was an antagonist for the PTH-2 receptor, but a full agonist for the PTH/PTHrP receptor. Residues 5 and 23 in PTH and PTHrP thus play key roles in signaling and binding interactions, respectively, with the PTH-2 receptor. Receptor-selective agonists and antagonists derived from these studies could help to identify the biological role of the PTH-2 receptor and to map specific sites of ligand-receptor interaction.     

Metabotropic glutamate receptor agonist (1S,3R-ACPD) increased frontal cortex dopamine release in aged but not in young rats

We examined the effects of inhibitors of calcium-calmodulin-dependent protein kinase II (CaM kinase II) and protein phosphatases on the glutamate (Glu) responses in cultured rat cerebellar Purkinje cells. CaM kinase II inhibitors significantly potentiated Glu responses, and activation of metabotropic Glu receptors facilitated this potentiation. In contrast, a phosphatase inhibitor calyculin A significantly reduced Glu responses. It was suggested that the Glu responsiveness of Purkinje cells may be regulated by the dynamic balance of phosphorylation and dephosphorylation of receptors or other relevant factors under basal conditions.     

2-substituted (2SR)-2-amino-2-((1SR,2SR)-2-carboxycycloprop-1-yl)glycines as potent and selective antagonists of group II metabotropic glutamate receptors. 2. Effects of aromatic substitution, pharmacological characterization, and bioavailability

In this paper we describe the synthesis of a series of alpha-substituted analogues of the potent and selective group II metabotropic glutamate receptor (mGluR) agonist (1S,1'S,2'S)-carboxycyclopropylglycine (2, L-CCG 1). Incorporation of a substitutent on the amino acid carbon converted the agonist 2 into an antagonist. All of the compounds were prepared and tested as a series of four isomers, i.e., two racemic diastereomers. On the basis of the improvement in affinity realized for the alpha-phenylethyl analogue 3, in this paper we explored the effects of substitution on the aromatic ring as a strategy to increase the affinity to these compounds for group II mGluRs. Affinity for group II mGluRs was measured using [3H]glutamic acid (Glu) binding in rat forebrain membranes. Antagonist activity was confirmed for these compounds by measuring their ability to antagonize (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid-induced inhibition of forskolin stimulated cyclic-AMP in RGT cells transfected with human mGluR2 and mGluR3. Meta substitution on the aromatic ring of 3 with a variety of substituents, both electron donating (e.g., methyl, hydroxy, amino, methoxy, phenyl, phenoxy) and electron withdrawing (e.g., fluorine, chlorine, bromine, carboxy, trifluoromethyl) gave from 1.5- to 4.5-fold increases in affinity. Substitution with p-fluorine, as in 97 (IC50 = 0.022 +/- 0.002), was the exception. Here, a greater increase in affinity was realized than for either the ortho- or meta-substituted analogues; 97 was the most potent compound resulting from monosubstitution of the aromatic. At best, only modest increases in affinity were realized for certain compounds bearing either two chlorines or two fluorines, and two methoxy groups gave no improvement in affinity (all examined in a variety of substitution patterns). Three amino acids, 4, 5, and 104, were resolved into their four constituent isomers, and affinity and functional activity for group II mGluRs was found to reside solely in the S,S,S-isomers of each, consistent with 1. With an IC50 = 2.9 +/- 0.6 nM, the resolved xanthylmethyl compound 168 was the most potent compound from this SAR. Amino acid 168 demonstrated high plasma levels following intraperitoneal (i.p.) administration and readily penetrated into the brain. This compound, however, had only limited (approximately 5%) oral bioavailability. Systemic administration of 168 protected mice from limbic seizures produced by the mGluR agonist 3,5-dihydroxyphenylglycine, with an ED50 = 31 mg/kg (i.p., 60 min preinjection). Thus, 168 represents a valuable tool to study the role of group II mGluRs in disease.     

Structural optimization affording 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4- (3-oxo-1,2,4-triazol-5-yl)methylmorpholine, a potent, orally active, long-acting morpholine acetal human NK-1 receptor antagonist

Structural modifications requiring novel synthetic chemistry were made to the morpholine acetal human neurokinin-1 (hNK-1) receptor antagonist 4, and this resulted in the discovery of 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4-(3-ox o-1 ,2,4-triazol-5-yl)methyl morpholine (17). This modified compound is a potent, long-acting hNK-1 receptor antagonist as evidenced by its ability to displace [125I]Substance P from hNK-1 receptors stably expressed in CHO cells (IC50 = 0.09 +/- 0.06 nM) and by the measurement of the rates of association (k1 = 2.8 +/- 1.1 x 10(8) M-1 min-1) and dissociation (k-1 = 0.0054 +/- 0.003 min-1) of 17 from hNK-1 expressed in Sf9 membranes which yields Kd = 19 +/- 12 pM and a t1/2 for receptor occupancy equal to 154 +/- 75 min. Inflammation in the guinea pig induced by a resiniferatoxin challenge (with NK-1 receptor activation mediating the subsequent increase in vascular permeability) is inhibited in a dose-dependent manner by the oral preadmininstration of 17 (IC50 (1 h) = 0.008 mg/kg; IC90 (24 h) = 1.8 mg/kg), indicating that this compound has good oral bioavailbility and peripheral duration of action. Central hNK-1 receptor stimulation is also inhibited by the systemic preadministration of 17 as shown by its ability to block an NK-1 agonist-induced foot tapping response in gerbils (IC50 (4 h) = 0.04 +/- 0.006 mg/kg; IC50 (24 h) = 0.33 +/- 0.017 mg/kg) and by its antiemetic actions in the ferret against cisplatin challenge. The activity of 17 at extended time points in these preclinical animal models sets it apart from earlier morpholine antagonists (such as 4), and the piperidine antagonists 2 and 3 and could prove to be an advantage in the treatment of chronic disorders related to the actions of Substance P. In part on the basis of these data, 17 has been identified as a potential clinical candidate for the treatment of peripheral pain, migraine, chemotherapy-induced emesis, and various psychiatric disorders.     

The group III metabotropic glutamate receptor agonist, l-AP4, reduces EPSPs in some layers of rat visual cortex

BACKGROUND: Vertical banded gastroplasty (VBG) is occasionally followed by poor weight loss or complications requiring reoperation. Several studies have analyzed the morbidity and mortality associated with conversions of VBG to gastric bypass, but few have described the actual technique. The most frequent complications related to this type of reoperation are gastrointestinal leaks. MATERIALS AND METHODS: The authors analyzed 60 consecutive conversions from VBG to lesser curvature gastric bypass, performed on 60 patients. The cases were analyzed for surgical technique, complications and weight loss. In all the cases the operation was limited to the lesser curvature of the stomach, and certain technical maneuvers were done to facilitate the creation of the pouch and anastomosis. RESULTS: There were three major complications, and two patients required reoperation. There were no gastrointestinal leaks or mortality. Percentage weight loss at 5 years was similar to primary gastric bypasses. CONCLUSION: Converting failed or complicated VBGs to lesser curvature gastric bypasses are safe and effective weight loss operations. By performing several specific technical maneuvers and limiting the operation to the highly vascular lesser curvature, complications can be reduced to a minimum.     

Synthesis of the selective 5-hydroxytryptamine 4 (5-HT4) receptor agonist (+)-(S)-2-chloro-5-methoxy-4-[5-(2-piperidylmethyl)-1,2,4-oxadiazol-3-y l]aniline

A 52-year-old white woman was first diagnosed with a tumor of the right optic nerve in 1972. She remained asymptomatic until 1992, when she had a seizure on the left side of her body from a frontoparietal glioblastoma multiforme. Ophthalmic examination revealed enlargement of the eye tumor. This case provides clinical documentation spanning 20 years of a growing, pigmented tumor of the optic nerve head shown histopathologically to be a retinal pigment epithelial adenoma.     

ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine]: I. A potent and selective cholinergic channel modulator with neuroprotective properties

Lactate dehydrogenase from the malarial parasite Plasmodium falciparum has many amino acid residues that are unique compared to any other known lactate dehydrogenase. This includes residues that define the substrate and cofactor binding sites. Nevertheless, parasite lactate dehydrogenase exhibits high specificity for pyruvic acid, even more restricted than the specificity of human lactate dehydrogenases M4 and H4. Parasite lactate dehydrogenase exhibits high catalytic efficiency in the reduction of pyruvate, kcat/Km = 9.0 x 10(8) min(-1) M(-1). Parasite lactate dehydrogenase also exhibits similar cofactor specificity to the human isoforms in the oxidation of L-lactate with NAD+ and with a series of NAD+ analogs, suggesting a similar cofactor binding environment in spite of the numerous amino acid differences. Parasite lactate dehydrogenase exhibits an enhanced kcat with the analog 3-acetylpyridine adenine dinucleotide (APAD+) whereas the human isoforms exhibit a lower kcat. This differential response to APAD+ provides the kinetic basis for the enzyme-based detection of malarial parasites. A series of inhibitors structurally related to the natural product gossypol were shown to be competitive inhibitors of the binding of NADH. Slight changes in structure produced marked changes in selectivity of inhibition of lactate dehydrogenase. 7-p-Trifluoromethylbenzyl-8-deoxyhemigossylic acid inhibited parasite lactate dehydrogenase, Ki = 0.2 microM, which was 65- and 400-fold tighter binding compared to the M4 and H4 isoforms of human lactate dehydrogenase. The results suggest that the cofactor site of parasite lactate dehydrogenase may be a potential target for structure-based drug design.     

2''-(Trimethylammonio)ethyl 4-(hexadecyloxy)-3(S)-methoxybutanephosphonate: a novel potent antineoplastic agent

The ability to successfully reuse OKT3, a mouse monoclonal antibody, is dependent upon the host's response to the antibody during and following the first treatment course. Antiidiotypic and/or antiisotypic antibodies may develop after exposure to OKT3. Antiidiotypic antibodies will bind OKT3, rendering it ineffective, while antiisotypic antibodies do not influence the efficacy of OKT3. A new membrane-based immunoassay, Transtat OKT3 (Sangstat Medical Corp, Menlo Park, CA) detects anti-OKT3 antibodies in less than 15 min. It allows simultaneous detection of antiidiotype and antiisotype antibodies. A total of 180 serum samples were initially analyzed by ELISA; results were negative, low-titer (1:100), or high-titer (> or = 1:1000). Retrospectively, these same samples were analyzed by Transtat for both anti-OKT3 (idiotype) and IgG2a (isotype). A total of 109 samples of 180 (60.6%) tested negative by ELISA and Transtat, while 71 (39.4%) tested positive. Of the negative samples by ELISA, 98 of 109 (89.9%) also tested anti-OKT3-negative by Transtat. Of the 109 specimens that were anti-OKT3 negative by Transtat, 98 (89.9%) tested negative by ELISA. There were 22 discrepant samples between the two methods; all were low-titer-positive (ELISA and Transtat). The 71 positive ELISA samples consisted of 53 low-titer (1:100) and 18 high-titer (> or = 1:1000), while the 71 anti-OKT3 positive Transtat samples consisted of 44 low-titer (1:10) and 27 high-titer (1:50). Sixty of 71 (84.5%) ELISA-positive samples were also positive by Transtat. Similarly, 60 of 71 (84.5%) Transtat-positive samples were also positive by ELISA. Of 71 patient samples positive for anti-OKT3 antibodies, 63 had an antiisotypic component present by Transtat. In conclusion, the Transtat OKT3 assay for measuring OKT3 and IgG2a antibodies offers a rapid and accurate assay for OKT3 monitoring.