Biomedical subjects

A M Stern

Publications and source records attributed to A M Stern.

At least 19 recordsLinked to original sources

PubMed1994

Transglutaminase inhibition by 2-[(2-oxopropyl)thio]imidazolium derivatives: mechanism of factor XIIIa inactivation.

The physiologic role of several transglutaminases could be more precisely defined with the development of specific inhibitors for these enzymes. In addition, specific plasma transglutaminase (fXIIIa) inhibitors may have therapeutic utility in the treatment of thrombosis. For these purposes, the inactivation of fXIIIa and human erythrocyte transglutaminase (HET) by 2-[(2-oxopropyl)thio]imidazolium derivatives, which comprise a novel class of transglutaminase inactivators, was studied. As a specific example, 1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride (III) inactivated fXIIIa with an apparent second-order rate constant (specificity constant of inactivation) of 6.3 x 10(4) M-1 s-1, corresponding to a rate 4 x 10(7) times greater than its reaction rate with glutathione (GSH). The mechanism of fXIIIa inactivation by this class of compounds was investigated utilizing two [14C]-isotopic regioisomers of 1,3-dimethyl-2-[(2-oxopropyl)thio]imidazolium iodide (II). Structural analyses demonstrated that acetonylation of the active site cysteinyl residue of fXIIIa occurred along with the stoichiometric release of the complementary fragment of the inactivator as the corresponding thione. Kinetic analysis of the inactivation of fXIIIa by nonquarternary analogs of II and III indicated the formation of a reversible complex between the inactivator and fXIIIa prior to irreversible modification of the enzyme. At 1 mM, III displayed no detectable levels of inhibition or inactivation with several serine proteases and thiol reagent-sensitive enzymes. 2-[(2-Oxopropyl)thio]imidazolium derivatives and the related molecule 2-(1-acetonylthio)-5-methylthiazolo-[2,3]-1,3,4-thiadiazo lium perchlorate (I), when present at the time of clot formation at 1-10 microM, enhanced the rates of tissue plasminogen activator catalyzed clot lysis in vitro. These inactivators prevented the fXIIIa-catalyzed covalent incorporation of alpha 2-antiplasmin into the alpha chain of fibrin and the formation of high molecular weight fibrin alpha chain polymers, providing the basis for the observed enhancements in clot lysis rates.

Animals↗

PubMed1994

A fully active catalytic domain of bovine aspartyl (asparaginyl) beta-hydroxylase expressed in Escherichia coli: characterization and evidence for the identification of an active-site region in vertebrate alpha-ketoglutarate-dependent dioxygenases.

The alpha-ketoglutarate-dependent dioxygenase aspartyl (asparaginyl) beta-hydroxylase (EC 1.14.11.16) specifically hydroxylates one aspartic or asparagine residue in certain epidermal growth factor-like domains of a number of proteins. The expression in Escherichia coli, purification, characterization of a fully active catalytic domain, and evidence for the identification of an active-site region of this enzyme are described. Sequence alignment analyses among the vertebrate alpha-ketoglutarate-dependent dioxygenases and chemical modification studies were undertaken aimed at locating specific regions of 52-kDa recombinant aspartyl (asparaginyl) beta-hydroxylase involved in substrate binding and/or catalysis. Based upon these studies, an alignment of the C-terminal regions of prolyl and lysyl hydroxylase and of aspartyl (asparaginyl) beta-hydroxylase is proposed. When histidine-675, an invariant residue located in a region of homology within this alignment, was mutated to an alanine residue in aspartyl (asparaginyl) beta-hydroxylase (H675A), no enzymatic activity was detected. Chemical modification studies show that the wild-type protein is protected from iodo[14C]acetamide labeling by Fe2+/alpha-ketoglutarate whereas the H675A mutant protein is not, suggesting that this mutant does not bind Fe2+/alpha-ketoglutarate.

Amino Acid Sequence↗

PubMed1993

Inhibition of HIV-1 reverse transcriptase by pyridinone derivatives. Potency, binding characteristics, and effect of template sequence.

The inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by pyridinone compounds has been investigated using as templates synthetic RNA with sequences based on the HIV-1 genome sequence. In reactions catalyzed by the enzyme that incorporated more than one nucleotide per primer, inhibition by a representative pyridinone inhibitor, 3-[2-(1,3-benzoxazol-2-yl)ethyl]-5-ethyl-6-methyl-pyridin-2(1H)one (L-696,229), was noncompetitive against deoxynucleotide triphosphate. For reactions that incorporated one deoxynucleotide per primer, IC50 values ranged from 20 to 200 nM, depending on the position of incorporation of the incoming deoxynucleotide base on the template. Inhibition of synthesis on a set of four templates differing only at the template base complementary to the incoming nucleotide had similar IC50 values. These results demonstrate that inhibitory potency is dependent on the primary structure of the template and that inhibitory potency is largely independent of the identity of the incoming nucleotide base. The inhibition of HIV-1 reverse transcription by L-696,229 also displayed slow-binding characteristics. The slow-binding aspect was exploited to gauge the interaction between inhibitor and enzyme. By titrating the reduction in the extent of the burst of synthesis observed in a reaction incorporating dideoxythymidine monophosphate into poly(rA)-oligo(dT)18, the apparent equilibrium constant for dissociation of the reverse transcriptase-L-696,229 complex was estimated to be 400 nM.

Antiviral Agents↗

PubMed1992

Invertebrate aspartyl/asparaginyl beta-hydroxylase: potential modification of endogenous epidermal growth factor-like modules.

An invertebrate alpha-ketoglutarate-dependent aspartyl/asparaginyl beta-hydroxylase, which posttranslationally hydroxylates specific aspartyl or asparaginyl residues within epidermal growth factor-like modules, was identified, partially purified and characterized. Preparations derived from two insect cell lines catalyzed the hydroxylation of the expected asparaginyl residue within a synthetic epidermal growth factor-like module. This activity was found to be similar to that of the purified mammalian aspartyl/asparaginyl beta-hydroxylase with respect to cofactor requirements, stereochemistry and substrate sequence specificity. Furthermore, recombinant human C1r, expressed in an insect cell-derived baculovirus expression system, was also found to be hydroxylated at the expected asparaginyl residue. Thus, these results establish the potential for invertebrate aspartyl/asparaginyl hydroxylation. Since several invertebrate proteins known to be required for proper embryonic development contain a putative consensus sequence that may be required for hydroxylation, the studies presented here provide the basis for further investigations concerned with identifying hydroxylated invertebrate proteins and determining their physiologic function.

Amino Acid Sequence↗

PubMed1992

cDNA cloning and expression of bovine aspartyl (asparaginyl) beta-hydroxylase.

Aspartyl (asparaginyl) beta-hydroxylase which specifically hydroxylates 1 Asp or Asn residue in certain epidermal growth factor-like domains of a number of proteins, has been previously purified to apparent homogeneity from detergent-solubilized bovine liver microsomes (Wang, Q., VanDusen, W. J., Petroski, C. J., Garsky, V. M., Stern, A. M., and Friedman, P. A. (1991) J. Biol. Chem. 266, 14004-14010). Three oligonucleotides, corresponding to three amino acid sequences of the purified hydroxylase, were used to screen bovine cDNA libraries. Several overlapping positive cDNA clones containing a full length open reading frame of 754 amino acids encoding a 85-kDa protein were isolated, and a cDNA, containing the full length open reading frame, was constructed from two of these clones. The resulting clone was then transcribed and translated in vitro to produce recombinant protein which possessed Asp beta-hydroxylase activity. These results constitute proof that the protein purified from bovine liver is an Asp beta-hydroxylase. Comparisons of deduced amino acid sequences of two other alpha-ketoglutarate-dependent dioxygenases, prolyl-4-hydroxylase and lysyl hydroxylase, with that of Asp beta-hydroxylase showed no significant homologies. Indeed, Asp beta-hydroxylase appears to be unique as no striking homology was found with known protein sequences. Furthermore, structural predictions derived from the deduced amino acid sequence are in accord with earlier Stokes' radius and sedimentation coefficient determinations of the enzyme, suggesting that the enzyme contains a relatively compact carboxyl-terminal catalytic domain and an extended amino terminus. This amino-terminal region has a potential transmembrane type II signal-anchor domain that could direct the catalytic domain into the lumen of the endoplasmic reticulum.

Amino Acid Sequence↗

PubMed1991

Tissue (type II) transglutaminase covalently incorporates itself, fibrinogen, or fibronectin into high molecular weight complexes on the extracellular surface of isolated hepatocytes. Use of 2-[(2-oxopropyl)thio] imidazolium derivatives as cellular transglutaminase inactivators.

Rabbit hepatocyte surface-expressed tissue (type II) transglutaminase is shown to act as a binding site for fibrinogen or fibronectin and to covalently incorporate these glycoproteins, in addition to itself, into extracellular high molecular weight complexes. This concept is supported by the observation that a nonpeptidyl, active site-directed transglutaminase inactivator (L683685) elicited concentration-dependent (0.1-10 microM) decreases in the calcium-dependent binding and covalent cross-linking of 125I-fibrinogen, 125I-fibronectin, or [14C]putrescine by hepatocyte suspensions. In corroboration with these findings, an antiserum against rabbit liver transglutaminase, which did not cross-react with rabbit factor XIII, elicited concentration-dependent decreases in the calcium-dependent binding and covalent cross-linking of 125I-fibrinogen or [14C]putrescine by hepatocyte suspensions. Western blots of sodium dodecyl sulfate/Triton-insoluble hepatocyte fractions conducted with this antiserum, with a polyclonal antiserum against human erythrocyte transglutaminase, or with a monoclonal antibody (CUB-7401) against guinea pig liver transglutaminase detected the 80-kDa tissue transglutaminase, as well as tissue transglutaminase-immunoreactive bands of higher molecular mass (range of 90 to greater than 200 kDa). The higher molecular weight species were preferentially incorporated, in a time- and calcium-dependent manner, into very high molecular weight complexes which did not enter the stacking gel. Incorporation of these tissue transglutaminase-containing bands into the high molecular weight complexes was inhibited by L683685, indicating that cross-linking by the enzyme was responsible for the assembly of the complexes of which tissue transglutaminase was itself a component. Cellular integrins did not mediate ligand binding under the experimental conditions, as evidenced by the failure of the Arg-Gly-Asp-Ser tetrapeptide or anti-integrin antibodies to inhibit binding or cross-linking of 125I-fibrinogen or 125I-fibronectin, in the presence or absence of transglutaminase inactivators.

Animals↗

PubMed1991

Bovine liver aspartyl beta-hydroxylase. Purification and characterization.

The alpha-ketoglutarate-dependent dioxygenase, L-asp(L-Asn)-beta-hydroxylase which posttranslationally hydroxylates specific aspartic acid (asparagine) residues within epidermal growth factor-like domains was purified from bovine liver and characterized. A 52-kDa and a 56-kDa species of this enzyme, which accounted for 60 and 30% of the total enzymatic activity, respectively, were purified to apparent homogeneity. Amino-terminal sequence analyses and immunoblots utilizing antisera raised to the intact 52-kDa species as well as to two complementary fragments of this species demonstrated that the 52- and 56-kDa species differ by a 22-amino acid amino-terminal extension. The remaining 10% of the purified enzymatic activity could be accounted for by the presence of immunologically related higher molecular mass forms (56-90 kDa) of L-Asp(L-Asn)-beta-hydroxylase. Strong evidence was obtained from the results of immunoextraction studies that L-Asp(L-Asn)-beta-hydroxylase can be identified with the purified proteins. Kinetic and physical studies suggest that L-Asp(L-Asn)-beta-hydroxylase exists as a monomer with a compact catalytic domain and an extended protease-sensitive amino terminus whose function remains to be determined. Since the purified L-Asp(L-Asn)-beta-hydroxylase hydroxylated both L-Asp- and L-Asn-containing substrates, it is possible that a single enzyme is responsible for the hydroxylation of Asp and Asn residues in vivo.

Amino Acid Sequence↗

PubMed1990

Enhanced thrombolysis by a factor XIIIa inhibitor in a rabbit model of femoral artery thrombosis.

Factor XIIIa (FXIIIa) catalyzes covalent crosslinking reactions of fibrin, affording the clot additional structural stability and resistance to plasmin-mediated degradation. Thus, inhibition of FXIIIa may render thrombi more susceptible to tissue-plasminogen activator (t-PA)-induced thrombolysis in vivo. We therefore examined thrombus weight and time to lysis in anesthetized rabbits undergoing arterial thrombosis produced by insertion of a copper coil into the lumen of the right femoral artery. The effects of t-PA alone (started 30 min after coil insertion) or in combination with a FXIIIa inhibitor (L722151) started 15 min before, 8 min after or 20 min after coil insertion were determined. Although t-PA alone (2 mg/kg over 2 hrs) lowered thrombus weight significantly, there was no evidence of flow restoration. Addition of L722151 to t-PA before, or 8 min after coil insertion, further lowered thrombus weights and produced thrombolysis in 50% of the animals. This beneficial effect was lost when L722151 administration was delayed until 20 min after thrombus formation, suggesting that the type(s) of crosslinking inhibited by L722151 was complete by this time. Infusion of L722151 alone had no significant effect on thrombus weight. These results demonstrate a time-dependent facilitation of t-PA-induced arterial thrombolysis by FXIIIa inhibition in a small animal model.

Animals↗

PubMed1990

Partial purification and characterization of bovine liver aspartyl beta-hydroxylase.

In vitro hydroxylation of aspartic acid has recently been demonstrated in a synthetic peptide based on the structure of the first epidermal growth factor domain in human factor IX (Gronke, R. S., VanDusen, W. J., Garsky, V. M., Jacobs, J. W., Sardana, M. K., Stern, A. M., and Friedman, P. A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3609-3613). The putative enzyme responsible for the posttranslational modification, aspartyl beta-hydroxylase, has been shown to be a member of a class of 2-ketoglutarate-dependent dioxygenases, which include prolyl-4- and lysyl-hydroxylases. In the present study, we describe the solubilization with nonionic detergent of the enzyme from bovine liver microsomes and its purification using DEAE-cellulose followed by heparin-Sepharose. No additional detergent was required during purification. The partially purified enzyme preparation was found to contain no prolyl-4- or lysyl-hydroxylase activity. Using a synthetic peptide based on the structure of the epidermal growth factor-like region in human factor X as substrate, the apparent Km values for iron and alpha-ketoglutarate were 3 and 5 microM, respectively. The enzyme hydroxylated the factor X peptide with the same stereospecificity (erythro beta-hydroxyaspartic acid) and occurred only at the aspartate corresponding to the position seen in vivo. Furthermore, the extent to which either peptide (factor IX or X) was hydroxylated reflected the extent of hydroxylation observed for both human plasma factors IX and X.

Amino Acid Sequence↗

PubMed1990

Inhibition of factor XIIIa in a canine model of coronary thrombosis: effect on reperfusion and acute reocclusion after recombinant tissue-type plasminogen activator.

The effect of inhibition of factor XIIIa with 2-(l-acetonylthio)-5-methylthiazolo[2,3-b]1,3,4-thiadiazo lium perchlorate (L-722,151) on coronary thrombolysis and reocclusion was studied in an acute dog model of electrically induced coronary thrombosis. L-722,151 (0.1 mg/kg/min intravenously [IV] or placebo was administered 15 minutes before current initiation (150 microA) and for the duration of the experiment (270 minutes). Fifteen minutes after thrombus formation, heparin (300 U/kg, IV) was administered, followed 45 minutes later by recombinant tissue-type plasminogen activator (tPA) (10 micrograms/kg/min, IV for 90 minutes). Placebo-treated animals thrombosed at 48.9 +/- 8.1 minutes (mean +/- SEM) and reperfused in response to tPA at 49.1 +/- 9.3 minutes. L-722,151 pretreated animals thrombosed at 44.4 +/- 9.7 minutes and reperfused in response to tPA at 16.4 +/- 2.8 minutes (P less than .05 v vehicle). Furthermore, residual thrombus mass was reduced by L-722,151 from 6.9 +/- 1.9 mg in placebo-treated animals to 1.7 +/- 0.6 mg (P less than .05 v vehicle). Acute reocclusion occurred in 86% of placebo and in 75% of L-722,151-treated animals. The incidence of tPA-induced reperfusion in L-722,151-treated dogs was 100% (8 of 8), whereas only 70% (7 of 10) of placebo-treated dogs reperfused. These results demonstrate that pretreatment with L-722,151 hastens reperfusion time threefold and reduces residual thrombus mass. These effects occurred with no change in systemic blood pressure in response to L-722,151. When L-722,151 was administered 15 minutes after thrombus formation in a separate group of dogs (n = 5), no beneficial effect on thrombolysis time or thrombus mass was observed. Thus, the specific factor XIIIa catalyzed crosslinking reaction(s), which may determine(s) resistance to plasmin-mediated fibrin degradation, occur(s) rapidly. Inhibition of this crosslinking by pretreatment with L-722,151 promotes tPA-induced thrombolysis.

Animals↗

PubMed1989

Preparation of a photoreactive analog of parathyroid hormone [Nle8, Lys(N-epsilon-4-azido-2-nitrophenyl)13,Nle18,Tyr34]bovine parathyroid hormone-(1-34)NH2, a selective, high-affinity ligand for characterization of parathyroid hormone receptors.

Photoaffinity radiolabeling techniques have been widely used to characterize the properties of peptide hormone receptors. However, the identity of authentic receptors is often uncertain because many macromolecules are labeled. These ambiguities are due, in part, to the use of a heterogeneous mixture of photoreactive photoligands, many of which have no or low affinity for the relevant hormone receptor. In this report, we describe the synthesis, purification, and structural analysis of the photoreactive parathyroid hormone analog, [Nle8,Lys(N-epsilon-4-azido-2-nitrophenyl)13,Nle18,Tyr34]-bovine parathyroid hormone-(1-34)NH2. The sulfur-free, oxidation-resistant, synthetic analog of bovine parathyroid hormone (PTH), [Nle8,Nle18,Tyr34]bovine PTH-(1-34)NH2 (NlePTH), was reacted with 4-fluoro-3-nitrophenylazide under nonaqueous conditions to yield several derivatives which were separated by reverse-phase high-performance liquid chromatography and analyzed by amino acid compositional analysis, thin-layer chromatography, and ultraviolet and visible absorption spectroscopy. Among the NlePTH derivatives generated, one of the least hydrophobic was shown to retain the highest potency as assessed in the canine renal cortical membrane radioreceptor assay. Sequence analysis of this peptide, after it had been derivatized with 4-fluoro-3-nitro-[2,6-3H]phenylazide and purified to homogeneity, permitted us to determine that the structure of this analog is [Nle8,Lys(N-epsilon-4-azide-2-nitrophenyl)13,Nle18,Tyr34]bovine PTH-(1-34)NH2. We emphasize the importance of using photoreactive ligands which are purified and subjected to detailed chemical and biological analyses for characterizing the properties of parathyroid hormone receptors and receptors for other peptide hormones.

Affinity Labels↗

PubMed1989

Aspartyl beta-hydroxylase: in vitro hydroxylation of a synthetic peptide based on the structure of the first growth factor-like domain of human factor IX.

beta-Hydroxylation of aspartic acid is a post-translational modification that occurs in several vitamin K-dependent coagulation proteins. By use of a synthetic substrate comprised of the first epidermal growth factor-like domain in human factor IX and either mouse L-cell extracts or rat liver microsomes as the source of enzyme, in vitro aspartyl beta-hydroxylation was accomplished. Aspartyl beta-hydroxylase appears to require the same cofactors as known alpha-ketoglutarate-dependent dioxygenases. The hydroxylation reaction proceeds with the same stereospecificity and occurs only at the aspartate corresponding to the position seen in vivo. Further purification and characterization of this enzymatic activity should now be possible.

Decarboxylation↗

PubMed1989

Peripheral metabolism of [35S]parathyroid hormone in vivo: influence of alterations in calcium availability and parathyroid status.

Parathyroid hormone (PTH) is rapidly metabolized, mainly by liver and kidney, to smaller fragments that are believed to be biologically inactive. The significance of this peripheral metabolism in the overall actions of PTH is unclear. Generation of circulating biologically active amino-terminal PTH fragments during metabolism in vivo has been suggested by certain observations in vitro, and what are believed to be amino-terminal fragments may be detectable in blood under pathological circumstances in vivo (such as renal failure and coexistent hyperparathyroidism) when highly sensitive assays are employed. We recently reported, however, that administration to normal rats of [35S]bovine PTH ([35S]bPTH) directly labelled at amino-terminal methionines, followed by high-resolution chromatographic analysis of extracted [35S]peptides, does not result in appearance of radioactive amino-terminal fragments in blood, even when the tracer is continuously infused to near-physiological plasma concentrations. We have now employed these techniques to address a second question regarding hormonal metabolism: is hormonal metabolism modified during metabolic perturbations such as changing calcium availability or altered levels of calciotrophic hormones? Metabolism of [35S-Met]bPTH (900 Ci/mmol), either alone or together with [3H-Pro]bPTH, however, did not lead to alterations in the rate of hormonal clearance nor to detectable circulating amino-terminal fragments, either in calcium-deprived or thyroparathyroidectomized rats or when animals were first rendered intoxicated with vitamin D or maintained on a high calcium intake. Likewise, tissue localization and specific cleavage patterns of intact hormone in liver or kidney were all unaltered by these various manoeuvres.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Peripheral metabolism of PTH: fate of biologically active amino terminus in vivo.

Clearance of intact parathyroid hormone (PTH) from blood is associated with rapid uptake by liver and kidney, limited proteolysis by tissue endopeptidases and, within minutes, appearance of circulating carboxyl-(COOH)-terminal PTH fragments. The fate of the corresponding amino(NH2)-terminal portion of the hormone during this peripheral metabolism is still unknown, however. To determine this, we have employed [35S]bovine PTH (bPTH) labeled to high specific activity at NH2-terminal methionines, which permits direct monitoring of the fate of the PTH NH2-terminus during metabolism in vivo. The [35S]PTH was administered by bolus or continuous intravenous infusion to anesthetized normal rats, to rats subjected to acute ablation of the liver, the kidneys, or both, and to rats receiving co-infusions of excess synthetic bPTH(1-34) NH2-terminal fragments. Analysis by high-resolution chromatographic techniques sensitive to 10(-13) M [35S]PTH peptides in plasma yields no evidence that peripheral metabolism of PTH generates circulating NH2-terminal fragments, even when special measures are taken to block clearance of such putative fragments from blood. We find that the NH2-terminus of PTH is rapidly degraded in situ by the liver but that both liver and especially kidney nevertheless contain low levels of NH2-terminal PTH fragments that, although not released into the blood, are large enough to be potentially active. Thus, the peripheral metabolism of PTH in normal animals does not normally lead to the formation of circulating amino terminal fragments of the hormone that might act independently of intact PTH on peripheral target tissues.

Animals↗

PubMed1987

Occurrence of beta-hydroxylated asparagine residues in non-vitamin K-dependent proteins containing epidermal growth factor-like domains.

Vitamin K-dependent bovine protein S has been shown to contain a posttranslationally hydroxylated asparagine within a conserved sequence in three of its epidermal growth factor (EGF)-like domains. In a review of amino acid sequences deduced from cDNA data, we have observed that a conserved sequence containing a potential asparagine hydroxylation site exists within EGF-like domains of a variety of functionally diverse proteins. We have studied a number of these and report the presence of erythro-beta-hydroxyasparagine (e-beta Hyn) in three non-vitamin K-dependent proteins: the plasma complement proteins C1r and C1s (where overbar indicates activated form) and the urinary protein uromodulin. For each protein, e-beta Hyn was identified in enzyme digests following the initial observation of erythro-beta-hydroxyaspartic acid (e-beta Hya) in acid hydrolysates of the proteins. e beta Hya and e-beta Hyn residues are detected by a postcolumn derivatization cation-exchange HPLC method herein described. HPLC isolation of the presumptive e-beta Hyn residue from enzyme digests of intact C1r allowed confirmation of its structure by GC/MS. Based upon available cDNA sequence data and observation of e-beta Hya in acid hydrolysates, we suggest other proteins in which e-beta Hyn may occur.

Asparagine↗

PubMed1985

Atrial overdrive pacing for conversion of atrial flutter in children.

Twenty-three successive patients with 27 different episodes of sustained atrial flutter were treated with atrial pacing for conversion of the tachyarrhythmia; 15 patients with 16 episodes of atrial flutter underwent intracardiac right atrial pacing and eight patients with 11 episodes of atrial flutter were treated with transesophageal atrial pacing. Ten of sixteen episodes (63%) and eight of 11 episodes (73%) were successfully converted using intracardiac and transesophageal techniques, respectively. Mean flutter cycle length for all 27 episodes was 219 ms (mean heart rate 274 beats per minute); successful pacing conversion cycle length (n = 15) was 72% of the flutter cycle length. Hemodynamic, electrophysiologic, and roentgenographic data were not predictive of conversion by either technique. Induction of localized atrial fibrillation or failure to meet critical pacing criteria may explain pacing failures. Based on this experience, a trial of transesophageal atrial pacing for acute conversion of any episode of atrial flutter in children prior to direct current cardioversion is recommended.

Adolescent↗

PubMed1984

Antitumor activity of DL-threo-beta-fluoroasparagine against human leukemia cells in culture and L1210 cells in DBA mice.

DL-threo-beta-Fluoroasparagine (DL-threo-beta-F-Asn) inhibited the growth of murine leukemia L1210 cells and three human leukemia cell lines in culture. Fifty % inhibiting dose values ranged between 30 and 50 microM DL-threo-beta-F-Asn. L1210 cells were not sensitive to DL-erythro-beta-fluoroasparagine, DL-threo-beta-fluoroaspartic acid, or DL-erythro-beta-fluoroaspartic acid at 300 microM, the highest dose studied. The antileukemia activity of DL-threo-beta-F-Asn was studied in further detail using the L1210 model system. Inhibition of growth in culture was prevented by L-asparagine but not by D-asparagine. Inhibition of growth of L1210 cells incubated for 40 hr in the presence of 300 microM DL-threo-beta-F-Asn was reversed after DL-threo-beta-F-Asn removal. Treatment for longer periods of time resulted in cell lysis. DL-threo-beta-F-Asn at doses of 250 mg/kg increased life span in mice bearing L1210 tumors by 60%. These results demonstrate the chemotherapeutic potential of the amino acid analogue DL-threo-beta-F-Asn.

Amino Acids↗

PubMed1983

DL-threo-beta-fluoroasparagine inhibits asparagine-linked glycosylation in cell-free lysates.

The effect of beta-fluoroasparagine on N-linked glycosylation was examined in a cell-free translation system in which glycosylation is coupled to protein synthesis. The threo-isomer markedly inhibited glycosylation at a concentration of 1 mM, and this effect was blocked by L-asparagine, indicating that glycosylation was inhibited secondary to incorporation of the asparagine analog into protein. The erythro-isomer, at similar concentrations, was not incorporated into protein and had no effect on glycosylation. threo-beta-Fluoroasparagine is highly toxic to some mammalian cells in culture. Our observations suggest that its toxicity may be due in part to the failure of the fluoroasparagine-containing protein to become glycosylated. The data suggest that this analog will be useful for examining the structural determinants for glycosylation.

Animals↗