Amino acid replacements resulting from suppression and missense reversion of a chain-terminator mutation in Neurospora.

The Neurospora crassa super-suppressor mutation, ssu-1, suppresses the auxotrophic phenotype of the mutant am(17) by inserting tyrosine at residue 313 of NADP-specific glutamate dehydrogenase, a position occupied in the wild type by glutamate. Two classes of am(17) revertants due to further mutation within the am gene have, respectively, tyrosine and leucine at residue 313. These replacements are consistent with a chain-terminating codon in am(17) of either the amber (UAG) or the ochre type (UAA), but are inconsistent with UGA. The Leu313 and Tyr313 variants of the enzyme have effective activity but are grossly different from the wild type in Michaelis constants (especially for ammonium) and heat stabilities at two different pH values. They show smaller but significant differences in these respects from each other.

Breast-milk composition in women with cystic fibrosis: report of two cases and a review of the literature.

Breast milk was collected from two women with cystic fibrosis (CF) and data from the literature was reviewed. The sodium concentration was within normal limits, 11-24 mmol/L in colostrum and 7-8 mmol/L in mature milk. Normal physiologic changes in milk composition after parturition (decreases in Na and increases in lactose) and during individual feeding periods (increases in fat and decreases in protein) were observed. Concentrations of milk protein, fat, and sugars were normal when the pulmonary disease of these patients was mild. During exacerbations of pulmonary disease, the concentrations of milk macronutrients were reduced. Milk secreted by women with CF appears to be physiologically normal, safe for the infant, and breast-feeding by mothers with CF should no longer be discouraged. Variations in the macronutrient content of CF milk warrants routine monitoring of the mother with CF and the breast-fed infant, especially during exacerbations in the pulmonary aspects of this disease.

2beta-Substituted analogues of 4'-iodococaine: synthesis and dopamine transporter binding potencies.

A series of 2beta-substituted analogues of 4'-iodococaine (3) was synthesized and evaluated in an in vitro dopamine transporter (DAT) binding assay. Selective hydrolysis at the 2beta-position of 3 gave the carboxylic acid 15 that served as the intermediate for the synthesis of compounds 4, 5, and 6-11. The 2beta-alkyl derivatives were obtained from ecgonine methyl ester (17) through a series of reactions leading to the aldehyde 20. Wittig reaction of 20 with methyltriphenylphosphorane followed by hydrogenation and benzoylation gave the products 12 and 13. The binding affinity of 4'-iodococaine (3) was 10-fold less than that of cocaine. The hydroxymethane, acetate, amide, benzyl ester, oxidazole, and ethane derivatives of 3 exhibited decreased binding while the vinyl, phenyl, and ethyl esters showed a moderate increase in binding affinity. Only the isopropyl derivative 8 exhibited a 2-fold increase in binding affinity compared with 4'-iodococaine (3). Hydroxylation of 8 at the 2'-position gave 14 which enhanced not only the binding potency at the DAT by another 2-fold but also the selectivity at the DAT over the norepinephrine and serotonin transporters. Compound 14 failed to stimulate locomotor activity in C57BL/6J mice over a wide dose range and blocked cocaine-induced locomotor stimulant action.

Synthesis and ligand binding studies of 4'-iodobenzoyl esters of tropanes and piperidines at the dopamine transporter.

Four analogs and two homologs of cocaine, designed as potent cocaine antagonists, were synthesized. The SN2 reaction between ecgonine methyl ester (13) or appropriately substituted piperidinol (19, 21) and appropriately substituted 4-iodobenzoyl chloride gave 4-iodobenzoyl esters of tropanes and piperidines (5-8). 2'-Hydroxycocaine (9) was obtained from 2'-acetoxycocaine (12) by selective transesterification with MeOH saturated with dry HCl gas. 2'-Acetoxycocaine (12) was synthesized from acetylsalicyloyl chloride (23) and ecgonine methyl ester (13). The binding affinities of these compounds were determined at the dopamine transporter for the displacement of [3H]WIN-35428. An iodo group substitution at the 4'-position of cocaine decreased dopamine transporter binding potency, while a hydroxy or acetoxy group at the 2'-position exhibited increased binding potency for the dopamine transporter compared to cocaine (10- and 3.58-fold, respectively). 2'-Hydroxylation also enhanced the bidning potency of 4'-iodococaine (5) by 10-fold. Replacement of the tropane ring with piperidine led to poor binding affinities.

The GABAA receptor alpha 6 subunit gene (Gabra6) is tightly linked to the alpha 1-gamma 2 subunit cluster on mouse chromosome 11.

We have established that the GABAA receptor alpha 6 (Gabra6) and alpha 1 (Gabra 1) subunit genes are tightly linked on mouse chromosome 11 by analysing the strain distribution patterns of RFLPs for the two genes and microsatellite markers flanking these genes in 26 BXD recombinant inbred strains. These results further demonstrate clustering of the GABAA receptor subunit genes on mouse chromosomes and the synteny for these clusters between the mouse and human genomes.

Anxiolytics by ethanol, diazepam and buspirone in a novel murine behavioral assay.

This study extends the pharmacological characterization of a novel quantitative murine behavioral method, the mirrored chamber aversion assay, which appears to be selectively sensitive to anxiolytic agents. Behavioral effects of acute diazepam administration were compared with those of the 5-HT1A anxiolytic buspirone and those of ethanol in C57BL/6J mice. These known anxiolytics produced a dose-dependent reduction in avoidance behavior of large magnitude, as evidenced by statistically significant decreases in latency to enter and increases in time spent in the mirrored chamber. Anxiolytic-like effects of these compounds in the mirrored chamber assay differed from those observed by the elevated plus-maze method. The behavioral effects of the test compounds were not due to alteration of locomotor activity. These findings indicate that the murine mirrored chamber assay responds to several agents known to be anxiolytic in man but differs from the plus-maze in the pharmacological spectrum of the anxiolytics to which it is sensitive.

Inherited, selective hyporesponsiveness to the analgesic action of nicotine in mice.

The acute dose-dependent analgesic activity of nicotine, as measured by the tail-flick assay, differed significantly between CD-1 and CF-1 outbred strains of mice. Differing responsiveness to the tail-flick stimulus did not explain this pharmacological effect. The inherent analgesic hyporesponsiveness of CF-1 mice was pharmacologically selective. Xilocaine and morphine produced an analgesic response of large magnitude in CF-1 mice. Reduced efficacy of nicotine in the CF-1 analgesia assay was not observed in its action on locomotor activity or in the induction of seizures and lethality. These findings have practical significance in identifying the importance of genotype in choice of strain for preclinical pharmacological studies of nicotine-induced analgesia and indicate that genetic analysis may provide a valuable tool for investigating the mechanism underlying the analgesic action of nicotine.

Inherited, selective hypoanalgesic response to cytisine in the tail-flick test in CF-1 mice.

This study extends the pharmacological characterization of the genotype- dependent difference in analgesic responsiveness to neuronal nicotinic agonists between CD-1 and CF-1 strains of mice. Acute analgesic potency of cytisine measured by the tail-flick assay differed by > 3200-fold between CD-1 and CF-1 outbred strains of mice. Analgesic non-responsiveness of the CF-1 strain was pharmacologically selective. Morphine produced a dose-dependent analgesic response of similar magnitude in both strains. Other pharmacological actions of cytisine, including inhibition of locomotor activity, induction of seizures and lethality, did not differ between these strains. Hyporesponsiveness to the analgesic action of both nicotine and cytisine was observed in two different CF-1 sublines. Biodistribution of [3H]cytisine in blood did not differ between the CF-1 and CD-1 strains. These pharmacological characteristics indicate that the CD-1-CF-1 strain pair provides a useful pharmacogenetic tool for investigating the mechanistic bases of analgesia induced by nicotinic cholinergic agonists.

2'-Substitution of cocaine selectively enhances dopamine and norepinephrine transporter binding.

Few studies have characterized the effect of substituents at the 2'-position of cocaine on transporter binding potency and selectivity. We synthesized 2'-OH-, 2'-F- and 2'-acetoxy-cocaines and compared their binding potencies for rat dopamine, norepinephrine and 5-hydroxytryptamine transporters to cocaine, 3'-OH-, 4'-OH-, 2'-OH,4'-I-cocaine derivatives, and to the transporter selective ligands WIN 35,428, nisoxetine and paroxetine. Unlike most substitutions, 2'-OH- and 2'-acetoxy-groups increased cocaine's binding potency for the dopamine transporter (10- and 4-fold, respectively). These substituents also enhanced binding to the norepinephrine transporter (52- and 35-fold, respectively) but had less effect on 5-hydroxytryptamine transporter binding. 2'-Hydroxylation also enhanced binding of 4'-I cocaine, an analog with low DA binding potency. The ability of 2'-substituents to substantially increase cocaine binding potency and to alter selectivity for brain transporters indicates the potential importance of the 2'-position in transporter binding.

Establishment of chronic intravenous drug self-administration in the C57BL/6J mouse.

A wide variety of drugs that have significant human abuse potential have been demonstrated to function as positive reinforcers in animals. The present study was designed to characterize a new mouse model of chronic intravenous drug self-administration. Adult male C57BL/6J mice, implanted with external jugular infusion catheters, were given access to response-contingent injections. They did not initiate responding for saline delivery, whereas the C57BL/6J mice initiated morphine, cocaine, methamphetamine and pentobarbital self-administration. Drug-maintained responding was consistently and significantly higher for each compound than for saline responding. In contrast to C57BL/6J mice, DBA/2J mice failed to initiate cocaine self-administration. Thus, chronic intravenous drug self-administration procedures can be adapted to the inbred mouse.

Selective behavioral alterations on addition of a 4'-phenyl group to cocaine.

We synthesized a cocaine analog in which a phenyl group was added at the para-position of the benzene ring of cocaine. This substitution caused a modest reduction (four-fold compared with cocaine) in binding potency for the primate (Papio) dopamine transporter as judged by displacement of [3H]WIN 35,428 binding from caudate/putamen membranes. Behavioral effects of this structural modification in the mouse were complex and selective, comprising absence of stimulation of locomotor activity, enhanced inhibition of locomotion and reduced lethal potency. Convulsant potency was unaltered. Substituents at the 4'-position of cocaine are important in its actions. Simple changes in the chemical structure of this drug may produce complex and selective changes in its neurochemical and behavioral actions.

Differential seizure sensitivities to picrotoxinin in two inbred strains of mice (DBA/2J and BALB/c ByJ): parallel changes in GABA receptor-mediated chloride flux and receptor binding.

Two strains of mice were shown to possess a differential sensitivity to picrotoxinin-induced convulsions; picrotoxinin elicited both tonic and clonic seizures at lower doses in the DBA/2J (DBA) strain compared to the BALB/c ByJ (BALB) strain. Less protection of picrotoxinin-induced tonic seizures was afforded by pentobarbital in the DBA strain. Biochemical studies revealed that picrotoxin inhibited 36Cl- efflux from forebrain synaptoneurosomes only in the DBA strain. In addition, picrotoxin inhibited pentobarbital-induced 36Cl- efflux to a greater extent in the DBA strain. No differences were observed in the binding of [3H]muscimol or [35S]t-butylbicyclophosphorothionate (TBPS) to forebrain homogenates, while pentobarbital was a less potent inhibitor of [35S]TBPS binding in the DBA strain. These findings suggest a genetic basis for the behavioral differences in convulsant sensitivity as well as for the neurochemical differences in allosteric coupling between convulsant and depressant/anticonvulsant sites associated with the GABA receptor-gated Cl- channel.

Sensitivity of inbred mice to methylxanthines is not determined by plasma xanthine concentration.

Male CBA/J and SWR/J mice were tested with doses of caffeine, theophylline and 8-p-sulfophenyltheophylline (xanthine). Caffeine produced dose-related decreases in locomotor activity and colonic temperatures in SWR/J mice. However, caffeine produced increases in locomotor activity and failed to lower the body temperature of CBA/J mice. Theophylline produced a decrease in body temperature of SWR/J mice. Comparison of brain caffeine levels demonstrated no difference in brain pharmacokinetics. The peripherally active xanthine failed to alter body temperature at the same molar dose as that of theophylline. These data clearly demonstrate that genetic differences in the effects of methylxanthine are due to inherent differences in the central nervous system sensitivity of the two strains. The data further indicate that while differences in xanthine metabolism may occur in inbred mice, these differences are not a major factor in the acute, peak plasma level, effects of xanthines.

Isolation and characterization of a polyamine-peptide conjugate from human amniotic fluid.

Significant amounts of the diamine putrescine and the polyamines spermidine and spermine could be detected in human third-trimester amniotic fluid only after acid hydrolysis. This observation was interpreted to mean that these amines existed only in conjugated form in this biological fluid. Upon fractionation by ultrafiltration 90--10% of the putrescine was associated with the 1000--10 000 dalton fraction. Spermine was identified in this fraction and in a low-molecular weight fraction presumably representing acetylated derivatives. Spermidine was entirely associated with the 10 000--30 000 dalton fraction. The putrescine conjugate was purified to homogeneity by column chromatography on Biogels P10 and P6 followed by ion-exchange chromatography on DEAE-Sephadex A-25. Molecular weight by gel exclusion using peptide standards was estimated to be approx. 4600. The UV absorption spectrum of the putrescine conjugate conformed to that expected for a polypeptide. This putrescine conjugate contained 39 identified amino acids with a combined molecular weight of 4713. Putrescine was detectable by high pressure liquid chromatography only after acid hydrolysis of the conjugate. No other polyamines were detected in these hydrolyzates, nor were any polyamines demonstable in hydrolyzates of control peptides nor in pooled column washes. The identity of the putrescine determined by high pressure liquid chromatography was confirmed by a two-dimensional thin-layer chromatography method. These results establish the in vivo production of a putrescine--polypeptide conjugate in man. Such molecular species may constitute yet another metabolic pathway for polyamines or may reflect another mode of post-translational modification of polypeptide structure and function. The qualitative and quantitative analysis of polyamine conjugate in human aminotic fluid may prove to be useful in the detection of abnormalities in fetal development.

Polyamine conjugates and total polyamine concentrations in human amniotic fluid.

A study of the quantitative profile of polyamines in amniotic fluid from the 13th through the 40th week of gestation was undertaken. These experimental observations indicate the absence of free putrescine, spermidine and spermine throughout gestation. Quantities of acid-liberated putrescine, spermidine and spermine are highest in late first and late third trimester. Putrescine is associated with peptide or peptides of molecular weight 1000 to 10 000 throughout gestation. Spermidine is found in amniotic fluid covalently conjugated to peptide or peptides with molecular weight 10 000 to 30 000. Spermine appears to exist in amniotic fluid, both in the higher molecular weight fraction (1000 to 10 000) and as acetylated derivatives. The existance of polyamine conjugates is compatible with an in vivo function in the regulation of embryonic growth and development. Abnormalities in polymines conjugated to peptides or their concentration may be useful in the diagnosis of fetal maldevelopment.

Plasma and submandibular saliva lysosomal enzymes in cystic fibrosis.

This study determined in a blind fashion the activity levels and thermostability properties of two lysosomal hydrolytic enzymes, acid phosphatase and alpha-mannosidase, in plasma samples from 25 cystic fibrosis (CF) patients and 25 age- and sex-matched normal controls. Mean alpha-mannosidase activity (3.2 +/- 1.0 mU/ml) and acid phosphatase activities (6.5 +/- 2.9 mU/ml) in CF patients were not significantly different from those found in normal individuals (2.8 +/- 0.7 and 7.6 +/- 3.4 mU/ml, respectively). Using stringent conditions no differences in thermostability properties of these enzymes were found between plasma from CF patients as compared to that of normal controls. When activity levels of these enzymes and of four additional hydrolytic enzymes, alpha-L-fucosidase, alpha-galactosidase, alpha-glucosidase and beta-galactosidase, were determined in submandibular saliva, no significant differences in enzyme levels between CF and age- and sex-matched controls were noted nor were thermostability differences found. Our data do not support the concept that altered properties of these enzymes are useful as markers for detection of CF homozygotes and heterozygotes, nor the hypothesis that the defect underlying this disease is a deficiency of post-translational modification of glycoproteins leading to their mis-compartmentalization and qualitative alteration.

Ontogeny of susceptibility to the convulsant, Ro 5-4864, and its relationship to audiogenic seizure susceptibility in inbred mice.

The postnatal development of susceptibility to the convulsant effects of Ro5-4864 (4'-chlorodiazepam) was characterized in two inbred mouse strains (DBA/2J and BALB/c ByJ) which as adults differ markedly in their response to this convulsant. Onset of susceptibility to a dose of Ro5-4864 which caused a high frequency of clonic seizures in adults was observed at 10 days of age in DBA/2 mice, but not until 35 days in BALB/c By mice. At 14 days of age an abrupt increase in susceptibility to Ro5-4864-induced tonic seizures was found in DBA/2 but not BALB/c By mice. Both the peak of tonic seizure susceptibility (21 days) and the time course of its subsequent age-dependent decline closely paralleled the change in audiogenic seizure susceptibility in the DBA/2 strain. PK11195 (40 mg/kg) blocked Ro5-4864 (25 mg/kg)-induced, age-dependent tonic seizures but had no effect on clonic seizure induction in the same mice. These observations establish that both the susceptibility to Ro5-4864 in adult mice and the postnatal time course for development of susceptibility to this convulsant are inherently different in these two strains of mice. The lack of coincidence between the developmental onset of susceptibility to Ro5-4864-induced seizures and the onset of supersensitivity to Ro5-4864-induced tonic seizures during the period of peak audiogenic seizure susceptibility in DBA/2 mice implies that more than one neurochemical mechanism is involved in the ability of Ro5-4864 to induce seizures in this strain. However, the blockade of Ro5-4864-induced tonic seizures by PK11195 suggests that peripheral type benzodiazepine receptors may mediate this effect.

3,7-Dimethyl-1-propargylxanthine: a potent and selective in vivo antagonist of adenosine analogs.

3,7-Dimethyl-1-propargylxanthine (DMPX), a caffeine analog that exhibits in vitro selectivity for A2-adenosine receptors, compared to A1-adenosine receptors, has now been investigated with respect to in vivo potency and selectivity. DMPX potently and selectively blocked the actions of the potent A2 adenosine agonist, 5'-N-ethylcarboxamidoadenosine (NECA), in DBA/2 mice, compared to blockade of the same responses elicited by the selective A1-adenosine agonist, N6-cyclohexyladenosine (CHA). DMPX was 57-fold more potent versus NECA-induced hypothermia than versus CHA-induced hypothermia and 11-fold more potent versus NECA-induced behavioral depression than versus CHA-induced behavioral depression. The hypothermia is mediated by peripheral receptors, based on blockade by 8-(p-sulfophenyl)theophylline (PSPT), while the behavioral depression is centrally mediated, based on lack of blockade by PSPT. DMPX was 28- and 15-fold more potent than caffeine in blocking peripheral and central NECA-responses, respectively. DMPX was equipotent with caffeine versus CHA-induced hypothermia and 2.5-fold more potent than caffeine versus CHA-induced behavioral depression. The motor stimulating potency of DMPX (ED50 10 mumol/kg) was slightly greater than caffeine.

Inherent differences in sensitivity to methylxanthines among inbred mice.

The behavioral effects of caffeine, theophylline, paraxanthine, and theobromine on locomotor activity were analyzed in four strains of inbred mice that were previously shown to differ in their acute toxic responses to caffeine administered at high dosages. Dose response curves for the effects of caffeine, theophylline, paraxanthine and theobromine on locomotor activity were established in CBA/J, C57BL/6J, DBA/2J and SWR/J strains of inbred mice. Paraxanthine was the maximally effective methylxanthine in the CBA/J, DBA/2J and SWR/J strains, while in the C57BL/6J strain, caffeine was the maximally effective methylxanthine. Theophylline failed to stimulate locomotor activity in the C57BL/6J strain and theobromine failed to stimulate activity in all of the strains tested. Decreases in locomotor activity were seen at the 100 mg/kg dose of caffeine in the C57BL/6J mice and at the 100 mg/kg dose of theophylline in the C57BL/6J, DBA/2J and SWR/J strains. Theobromine produced decreases in locomotor activity in the C57BL/6J, DBA/2J and SWR/J strains of mice. In contrast to the other methylxanthines, paraxanthine failed to decrease activity across the range of doses tested (1.0-150 mg/kg). These data suggest that the methylxanthines have genetically specified multiple modes of action upon locomotor activity and that the use of genetically distinct strains of mice may have important value in the neurochemical and pharmacological dissection of methylxanthine-induced behavioral effects.

Effects of chronic amphetamine in BALB/cBy mice, a strain that is not stimulated by acute administration of amphetamine.

The effects of d-amphetamine and methylphenidate on locomotor activity of BALB/cByJ mice were evaluated. d-Amphetamine had no effect or inhibited locomotor activity at acute doses of up to 10 mg/kg while methylphenidate stimulated locomotor activity at acute doses between 10 and 32 mg/kg. The dose-response curves for methylphenidate and d-amphetamine appeared to be quantal in nature. During a 21-day chronic treatment with 10 mg/kg d-amphetamine no evidence of tolerance to the depressant effects of relatively high doses of d-amphetamine was observed. However, a 3.2 mg/kg dose of d-amphetamine, which acutely inhibited locomotor activity, was found to stimulate locomotor activity following chronic amphetamine treatment. Doses of methylphenidate which acutely stimulated activity were without effect in mice chronically receiving amphetamine. Although the mechanism underlying these behavioral effects has yet to be established, our results indicate that inherent alterations can differentially affect both acute and chronic susceptibility to the behavioral effects of amphetamine and methylphenidate. Use of such altered strains of mice can be especially revealing of subtle behavioral effects brought about by chronic drug treatment which are not readily demonstrated following acute administration of amphetamine.