Histological and computed tomographic evaluation of a parasitic conjoined twin in hybrid catfish (Ictalurus punctatus [Rafinesque] X Ictalurus furcatus [Lesueur]).

There is growing use of hybrid catfish (Ictalurus punctatus ♀ X Ictalurus furcatus ♂) in commercial aquaculture to utilize hybrid vigour to improve production A conjoined twin specimen found during the course of production studies by the United States Department of Agriculture Catfish Genetic Research Unit (USDA-CGRU) was submitted to the Aquatic Research and Diagnostic Laboratory (ARDL). After preliminary inspection, it was transported to Mississippi State University, College of Veterinary Medicine for further evaluation. The specimen was examined using both computed radiography and computed tomography antemortem. Following humane euthanasia, the specimen was examined both grossly and histologically. Tissues from both fish were also submitted for genetic analysis to determine whether twins were derived from the same egg. This report records the presentation and examination of a pair of conjoined hybrid catfish (I. punctatus X Ictalurus furcatus).

Reaction sequences for (Na+ + K+)-dependent ATPase hydrolytic activities: new quantitative kinetic models.

To delineate better the reaction sequence of the (Na+ + K+)-ATPase and illuminate properties of the active site, kinetic data were fitted to specific quantitative models. For the (Na+ + K+)-ATPase reaction, double-reciprocal plots of velocity against ATP (in the millimolar range), with a series of fixed KCl concentrations, are nearly parallel, in accord with the ping pong kinetics of ATP binding at the low-affinity sites only after Pi release. However, contrary to requirements of usual formulations, Pi is not a competitor toward ATP. A new steady-state kinetic model accommodates these data quantitatively, requiring that under usual assay conditions most of the enzyme activity follows a sequence in which ATP adds after Pi release, but also requiring a minor alternative pathway with ATP adding after K+ binds but before Pi release. The fit to the data also reveals that Pi binds nearly as rapidly to E2 X K X ATP as to E2 X K, whereas ATP binds quite slowly to E2 X P X K: the site resembles a cul-de-sac with distal ATP and proximal Pi sites. For the K+-nitrophenyl phosphatase reaction also catalyzed by this enzyme, the apparent affinities for both substrate and Pi (as inhibitor) decrease with higher KCl concentrations, and both Pi and TNP-ATP appear to be competitive inhibitors toward substrate with 10 mM KCl but noncompetitive inhibitors with 1 mM KCl. These data are accommodated quantitatively by a steady-state model allowing cyclic hydrolytic activity without obligatory release of K+, and with exclusive binding of substrate vs. either Pi or TNP-ATP. The greater sensitivity of the phosphatase reaction to both Pi and arsenate is attributable to the weaker binding by the occluded-K+ enzyme form occurring in the (Na+ + K+)-ATPase reaction sequence. The steady-state models are consistent with cyclical interconversion of high- and low-affinity substrate sites accompanying E1/E2 transitions, with distortion to low-affinity sites altering not only affinity and route of access but also separating the adenine- and phosphate-binding regions, the latter serving in the E2 conformation as the active site for the phosphatase reaction.

Cation sites, spermine, and the reaction sequence of the (Na+ + K+)-dependent ATPase.

Spermine, at 0.3 mM, inhibits the K+-nitrophenyl phosphatase activity of a dog kidney (Na+ + K+)-ATPase preparation, increasing the K0.5 for K+, reducing the Km for substrate, and affecting little the inhibition by Na+. These actions can be attributed, in a model of the phosphatase reaction, to parallel decreases in affinity for K+ and Na+ at their cytoplasmically accessible sites. In the (Na+ + K+)-ATPase reaction, spermine increases the K0.5 for Na+ and, to a lesser degree, the K0.5 for K+ as activators. With spermine, the double-reciprocal plots of velocity vs. ATP concentration (in the range 0.3-3 mM), at fixed levels of K+ (from 1 to 10 mM), remain parallel but are rotated clockwise and spread somewhat, reflecting stimulation at low ATP concentrations and inhibition at high ATP but low KCl concentrations. These actions can be attributed, in a steady-state ping-pong model of the ATPase reaction, solely to decreased rates of binding of Na+ and K+ to their sites, with major effects at the cytoplasmically accessible sites for Na+ (acceptance) and K+ (discharge), and with a lesser effect at the extracellularly accessible sites for K+ (acceptance). On these grounds, spermine is a highly specific and potentially valuable reagent for studying the reaction. Furthermore, the model for K+-ATP interactions not only supports a specific reaction sequence (K+ addition, Pi release, ATP addition, K+ release) but also argues against the availability of low-affinity substrate sites except during sharply restricted segments of the reaction sequence, thereby favoring proposals that the low-affinity substrate sites are transformed into high-affinity substrate sites with the E2 to E1 conformational change.

Reversible inhibitors of the gastric (H+/K+)-ATPase. 2. 1-Arylpyrrolo[3,2-c]quinolines: effect of the 4-substituent.

Further work on compounds 1 has identified the 4-position as a site where substantial modifications are tolerated, leading to analogues which are more potent and less toxic than those described previously. The best compound in the series is 13a (SK&F 96356), which is a potent inhibitor of gastric acid secretion in both the pentagastrin-stimulated rat and the histamine-stimulated dog. This compound shows reversible, K(+)-competitive binding to the enzyme. Because of its fluorescent properties, it is also proving useful in vitro as a probe of the structure and function of the (H+/K+)-ATPase.

Reversible inhibitors of the gastric (H+/K+)-ATPase. 4. Identification of an inhibitor with an intermediate duration of action.

3-Acyl-4-(arylamino)quinolines were previously identified as gastric (H+/K+)-ATPase inhibitors, and clinical efficacy has been demonstrated for compound 3 (SK&F 96067). In the present study the further structure-activity relationship of this series is developed. Only a limited range of substituents are tolerated on the N-aryl ring or the 6- and 7-positions of the quinoline, and although hydroxylated derivatives were identified possessing markedly greater affinity for the enzyme, none of these proved to have adequate potency after oral dosing. In contrast, the 8-position of the quinoline ring proved suitable for a wide variety of substituents, allowing modification of physicochemical properties while retaining primary activity. This led to the identification of compound 4 (SK&F 97574), which combines good oral potency with a somewhat longer duration of action than 3 (though much shorter than covalent inhibitors such as omeprazole). This compound was selected for further development and evaluation in man.

Reversible inhibitors of the gastric (H+/K+)-ATPase. 5. Substituted 2,4-diaminoquinazolines and thienopyrimidines.

Quinazolines bearing a secondary 4-(arylamino) substituent demonstrate an SAR for inhibition of the gastric (H+/K+)-ATPase different from the previously described 3-acylquinolines, suggesting that, although these compounds are also K(+)-competitive, they probably bind to the enzyme in a different orientation. Compounds bearing a tertiary 4-(arylamino) substituent, however, in particular 4-(N-methylarylamino), appear to possess an SAR quite similar to the 3-acylquinolines. We show that this arises from the effect of the N-methylation, which is to orientate the 4-(arylamino) substituent syn to C5, analogous to the 3-acylquinolines. Compounds possessing both a 4-(N-methylarylamino) substituent and a 2-(arylamino) substituent proved to be very potent, K(+)-competitive inhibitors of K(+)-stimulated ATPase activity with Ki values down to 12 nM. Some compounds also proved to be effective inhibitors of stimulated acid secretion in both the rat and dog when dosed intravenously. However, although a number of these demonstrated activity after oral administration in the dog, the level and variability precluded further evaluation.

Reversible inhibitors of the gastric (H+/K+)-ATPase. 1. 1-Aryl-4-methylpyrrolo[3,2-c]quinolines as conformationally restrained analogues of 4-(arylamino)quinolines.

The 4-(arylamino)quinoline 4, previously described as an antiulcer compound, is shown to be an inhibitor of the gastric (H+/K+)-ATPase. It is postulated that 1-arylpyrrolo[3,2-c]quinolines 6 act as conformationally restrained analogues of 4. A series of derivatives of 6 has been prepared and shown to be potent inhibitors of the target enzyme in vitro. Substitution in the ortho position of the aryl ring is important for activity. Unsaturation in the 5-membered ring makes little difference, but introduction of heteroatoms into the same ring markedly reduces activity. In more detailed kinetic experiments, 15c and 4 both show reversible, K(+)-competitive binding to the enzyme, with submicromolar Ki values. The compounds appear to act at the lumenal face of the enzyme and to require protonation for activity. Several compounds in the series are shown to be potent inhibitors of pentagastrin-stimulated acid secretion in the rat.

Reversible inhibitors of the gastric (H+/K+)-ATPase. 3. 3-substituted-4-(phenylamino)quinolines.

Previously, gastric (H+/K+)-ATPase inhibitors such as 2 have been prepared as analogues of 1a on the presumption that the 3-carbethoxy substituent plays a key role in establishing the orientation of the 4-arylamino group. In this paper we explore further the contribution made to activity by the quinoline 3-substituent. We show that, for compounds bearing such a substituent, only a particular combination of properties provides high activity, both in vitro and as inhibitors of gastric acid secretion in vivo. The ability of the substituent to affect activity by restricting rotation about the Cquin-N bond through a combination of both a pi-electron withdrawal and hydrogen bonding is supported by the current study. However, high activity is only achieved if the effect of this group on the quinoline pK(a) is kept to a minimum. 3-Acyl substituents provide an optimum combination of electronic properties. From this series, compound 17c (SK&F 96067) was shown to be a potent inhibitor of histamine-stimulated gastric acid secretion after oral dosing in the Heidenhain pouch dog and was selected for further development and evaluation in man.

SK&F 96067 is a reversible, lumenally acting inhibitor of the gastric (H+ + K+)-ATPase.

SK&F 96067 [3-butyryl-4-(2-methylphenylamino)-8-methoxyquinoline] has been identified, from a novel class of 4-aminoquinolines, as a reversible inhibitor of the gastric (H+ + K+)-ATPase. This compound has been studied in gastric membrane vesicle preparations enriched in the (H+ + K+)-ATPase. At pH 7.0, SK&F 96067 inhibited K(+)-stimulated ATPase activity competitively with respect to the activating cation K+, with a Ki value of 0.39 +/- 0.05 microM. Under comparable conditions, SK&F 96067 was 32 times more potent as an inhibitor of the gastric (H+ + K+)-ATPase relative to the closely related (Na+ + K+)-ATPase. Studies in intact gastric vesicles showed that SK&F 96067 also inhibited hydrogen ion transport. Using the initial rate of acridine orange quenching as the index of acidification, an IC50 of 0.84 +/- 0.24 microM was observed. Steady state acidification, as measured by aminopyrine accumulation, was inhibited with greater potency (IC50 = 0.06 +/- 0.01 microM) consistent with the accumulation of this inhibitor into the intravesicular acidic space to a site of action on the inside (lumenal) face of the enzyme. Inhibition of ATPase activity in the presence of both SK&F 96067 and the K(+)-competitive (H+ + K+)-ATPase inhibitor, SCH 28080, indicated that their binding was mutually exclusive, consistent with SK&F 96067 acting at the same lumenal binding site as does SCH 28080. The steady-state inhibition kinetics of SK&F 96067 against K(+)-stimulated ATPase activity were followed as a function of pH. At pH 6.6 and 7.0 the inhibition was competitive with respect to the activating cation K+. At pH 7.5 and 8.1 a mixed pattern of inhibition was detected. Thus, at alkaline pH values, the binding of SK&F 96067 and K+ were no longer mutually exclusive. The potency of SK&F 96067 decreased as pH rose, consistent with the protonated form of the inhibitor being the preferred inhibitory species. A kinetic model is discussed, in which, at acidic pH, the protonated form of SK&F 96067 binds to the enzyme competitively with respect to K+, whereas, at alkaline pH, the neutral form of SK&F 96067 can bind simultaneously with K+.

Hypernickelemia following coronary arteriography, caused by nickel in the radiographic contrast medium.

Meglumine diatrizoate ("Renografin-76", a radiographic contrast medium) contains sufficient nickel to cause hypernickelemia in patients after coronary arteriography. Nickel analyses by electrothermal atomic absorption spectrophotometry showed that nine lots of "Renografin-76" (760 g of meglumine diatrizoate per L) contained 144 +/- 44 micrograms Ni per L. Serum Ni concentrations became elevated in 11 patients after coronary arteriography (Ni dose = 19 +/- 4 micrograms per patient); peak Ni concentrations (increment = 1.8 +/- 0.4 micrograms Ni per L) occurred 0.25 or 0.5 h post-injection. Serum Ni concentrations diminished at 2 and 4 h post-injection and returned to base-line values at 24 h. The half-time (T1/2) for reduction of serum Ni concentrations averaged 1.5 h. Analysis of urine specimens from two patients showed that most of the Ni dose was excreted in urine within 24 hours. After iv administration of meglumine diatrizoate to rabbits (0.5 or 1.0 micrograms Ni per kg body wt), T1/2 values for elimination of Ni from the serum volume averaged 1.2 h, compared to T1/2 values of 5.7 and 7.4 h, respectively, when Ni was administered iv in NiCl2 or albumin solutions. Since "Renografin-76" contains edetate disodium (0.4 g per L), Ni is probably present as a Ni-EDTA complex, accounting for the rapid elimination of Ni following iv administration of the contrast medium to patients and rabbits. To reduce possible hazards of allergic or cardiovascular reactions to nickel, the authors recommend that Ni concentrations in radiographic contrast media should not exceed 10 micrograms per L.

Lipoprotein-associated PLA2 inhibition--a novel, non-lipid lowering strategy for atherosclerosis therapy.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a serine lipase that is associated with low density lipoprotein (LDL) in human plasma. Substrates include oxidised phosphatidylcholine (PC), which is hydrolysed by Lp-PLA2 to lyso-PC and oxidised fatty acids. Both products are bioactive and proinflammatory, and implicated in monocyte infiltration into the developing plaque, deposition of foam cells, and plaque progression and instability. Lp-PLA2 has recently been shown to be a risk factor for coronary events in previously asymptomatic, hypercholesterolaemic men. A series of azetidinones was designed as potent and selective inhibitors of this enzyme; SB-222657 inhibited release of the chemotactic cleavage products from oxidised LDL, and SB-244323 reduced atherosclerotic plaque development in a 3 month rabbit study. A series of pyrimidones has been designed from a screening hit, and nanomolar inhibitors identified. Oral efficacy in inhibiting plasma Lp-PLA2 in rabbits has been demonstrated with a variety of structural classes.

GSK1562590, a slowly dissociating urotensin-II receptor antagonist, exhibits prolonged pharmacodynamic activity ex vivo.

BACKGROUND AND PURPOSE: Recently identified antagonists of the urotensin-II (U-II) receptor (UT) are of limited utility for investigating the (patho)physiological role of U-II due to poor potency and limited selectivity and/or intrinsic activity. EXPERIMENTAL APPROACH: The pharmacological properties of two novel UT antagonists, GSK1440115 and GSK1562590, were compared using multiple bioassays. KEY RESULTS: GSK1440115 (pK(i)= 7.34-8.64 across species) and GSK1562590 (pK(i)= 9.14-9.66 across species) are high affinity ligands of mammalian recombinant (mouse, rat, cat, monkey, human) and native (SJRH30 cells) UT. Both compounds exhibited >100-fold selectivity for UT versus 87 distinct mammalian GPCR, enzyme, ion channel and neurotransmitter uptake targets. GSK1440115 showed competitive antagonism at UT in arteries from all species tested (pA(2)= 5.59-7.71). In contrast, GSK1562590 was an insurmountable UT antagonist in rat, cat and hUT transgenic mouse arteries (pK(b)= 8.93-10.12 across species), but a competitive antagonist in monkey arteries (pK(b)= 8.87-8.93). Likewise, GSK1562590 inhibited the hU-II-induced systemic pressor response in anaesthetized cats at a dose 10-fold lower than that of GSK1440115. The antagonistic effects of GSK1440115, but not GSK1562590, could be reversed by washout in rat isolated aorta. In ex vivo studies, GSK1562590 inhibited hU-II-induced contraction of rat aorta for at least 24 h following dosing. Dissociation of GSK1562590 binding was considerably slower at rat than monkey UT. CONCLUSIONS AND IMPLICATIONS: Whereas both GSK1440115 and GSK1562590 represent high-affinity/selective UT antagonists suitable for assessing the (patho)physiological role of U-II, only GSK1562590 exhibited sustained UT residence time and improved preclinical efficacy in vivo.

A K+ competitive, conformational probe of the H,K-ATPase.

The H,K-ATPase was noncovalently labelled with a fluorescent quinoline derivative, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo [3,2-c]quinoline, (MDPQ). MDPQ competitively inhibited the K+ stimulated ATP hydrolysis with a Ki of 0.22 microM but did not inhibit the MgATP-dependent phosphoenzyme to an extent greater than 10% of control. Inhibitor binding to the H,K-ATPase enhanced MDPQ fluorescence. This fluorescence was quenched by lumenal K+ with a K0.5 of 1.8 mM. MDPQ binding to the H,K-ATPase shifted the fluorescence Ex/Em maxima from 342/478 nm to 342/453 nm. Phosphorylation of the H,K-ATPase by MgATP further enhanced fluorescence with a difference spectra [MgATP-(MgATP+KCl)] emission peak at 446 nm. Trypsin dependent proteolysis of the H,K-ATPase stabilized within the E2K conformation eliminated the phosphoenzyme response, but enhanced the K+ specific dephosphoenzyme response. These observations show that MDPQ is a fluorescent, competitive inhibitor of the H,K-ATPase that interacts with a lumenal cation binding site. Under specific conditions, both the cation and MDPQ binding sites remain intact within trypsin produced cleavage peptides of the H,K-ATPase.

Mechanism of inhibition of LDL phospholipase A2 by monocyclic-beta-lactams. Burst kinetics and the effect of stereochemistry.

Investigation of the inhibition of LDL-associated phospholipase A2 by monocyclic beta-lactams has shown that LDL phospholipase A2 is capable of hydrolyzing monocyclic-beta-lactams by a mechanism which shares many similarities to the hydrolysis of beta-lactams by beta-lactamases. We believe that this is the first demonstration of a serine-dependent lipase being able to hydrolyze an amide bond. Although 4-(phenylthio)-N-(4-phenyl-2-oxobutyl)azetidin-2-one, SB-216477, and its enantiomers are relatively modest covalent inactivators with kobs/[I] = 46 M-1 s-1 for the R enantiomer, analysis of the kinetics of inactivation and reactivation shows that these compounds act as slow-turnover substrates, presumably via an acylation-deacylation mechanism. The detection of a suprastoichiometric burst indicates that the pathway must be branched with the branching giving rise to the slow reactivation via a more stable covalent intermediate. Study of the two enantiomers of SB-216477 shows that LDL-associated phospholipase A2 is sensitive to the beta-lactam stereochemistry at C4. However, a common achiral intermediate is formed along the turnover pathway, and this must be at or immediately prior to the branch point.

Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor.

A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki=40+/-3 nM, kobs/[I]=6. 6x10(5) M-1.s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0+/-0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki=6.3+/-0.5 microM, kobs/[I]=1.6x10(4) M-1.s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine.

The identification of a potent, water soluble inhibitor of lipoprotein-associated phospholipase A2.

Modification of the pyrimidone 5-substituent in a series of 1-((amidolinked)-alkyl)-pyrimidones, lipophilic inhibitors of lipoprotein-associated phospholipase A2, has given inhibitors of nanomolar potency and improved physicochemical properties. Compound 23 was identified as a potent, highly water soluble. CNS penetrant inhibitor suitable for intravenous administration.

1-(Arylpiperazinylamidoalkyl)-pyrimidones: orally active inhibitors of lipoprotein-associated phospholipase A(2).

The lipophilic 1-substituent in a series of 1-((amidolinked)-alkyl)-pyrimidones, inhibitors of recombinant lipoprotein-associated phospholipase A(2), has been modified to give inhibitors of high potency in human plasma and enhanced physicochemical properties. Phenylpiperazineacetamide derivative 23 shows very promising oral activity.

N-1 substituted pyrimidin-4-ones: novel, orally active inhibitors of lipoprotein-associated phospholipase A2.

From two related series of 2-(alkylthio)-pyrimidones, a novel series of 1-((amidolinked)-alkyl)-pyrimidones has been designed as nanomolar inhibitors of human lipoprotein-associated phospholipase A2. These compounds show greatly enhanced activity in isolated plasma. Selected derivatives such as compounds 51 and 52 are orally active with a good duration of action.

2-(Alkylthio)pyrimidin-4-ones as novel, reversible inhibitors of lipoprotein-associated phospholipase A2.

Starting from two weakly active hits from high throughput screening, a novel series of 2-(alkylthio)-pyrimidin-4-ones with high potency and selectivity for lipoprotein-associated phospholipase A2 has been designed. In contrast to previously known inhibitors, these have been shown to act by a non-covalent and substrate competitive mechanism.