MM Isoenzyme Of Creatine Kinase Research Articles

Study on newborn screening for Duchenne muscular dystrophy and diagnostic strategy

To establish a newborn screening system for Duchenne muscular dystrophy (DMD) through assessment of MM isoenzyme of creatine kinase (CK-MM) activity. The CK-MM level was detected using dry blood spot filter paper from 10 252 male newborns. The results were grouped based on their gestational age, sampling time and intervals between the experiments. The threshold value for CK-MM necessitating genetic testing was determined. Next-generation sequencing (NGS) was carried out for those with a CK-MM value over the threshold, and the result was verified by multiplex ligation-dependent probe amplification (MLPA). Based on the result of non-parametric rank sum test, the median CK-MM concentration has increased with the gestational age, and was inversely correlated with the age of the newborns among unaffected specimens. CK-MM on dry blood spot filter paper can be stable for 14 days at 2-8℃. Statistical analysis of CK-MM value of the 10 252 neonates suggested that the threshold may be set as 700 ng/mL. Exonic deletions were found in 2 confirmed cases, whose CK-MM level was greater than 2000 ng/mL. Detection of CK-MM in dry blood spot filter paper has provided an effective method for newborn screening of DMD. This simple and inexpensive method can be used for large-scale screening, which is of great value to the early intervention and treatment of the disease.

MENS-associated increase of muscular protein content via modulation of caveolin-3 and TRIM72.

Microcurrent electrical neuromuscular stimulation (MENS) is known as an extracellular stimulus for the regeneration of injured skeletal muscle in sports medicine. However, the effects of MENS-associated increase in muscle protein content are not fully clarified. The purpose of this study was to investigate the effects of MENS on the muscular protein content, intracellular signals, and the expression level of caveolin-3 (Cav-3), tripartite motif-containing 72 (TRIM72) and MM isoenzyme of creatine kinase (CK-MM) in skeletal muscle using cell culture system. C2C12 myotubes on the 7th day of differentiation phase were treated with MENS (intensity: 10-20 microA, frequency: 0.3 Hz, pulse width: 250 ms, stimulation time: 15-120 min). MENS-associated increase in the protein content of myotubes was observed, compared to the untreated control level. MENS upregulated the expression of Cav-3, TRIM72, and CK-MM in myotubes. A transient increase in phosphorylation level of Akt was also observed. However, MENS had no effect on the phosphorylation level of p42/44 extracellular signal-regulated kinase-1/2 and 5'AMP-activated protein kinase. MENS may increase muscle protein content accompanied with a transient activation of Akt and the upregulation of Cav-3 and TRIM72.

急性心筋梗塞症にみられたCreatine Kinase Mサブユニット欠損症の免疫学的研究

急性心筋梗塞症にみられたCreatine Kinase Mサブユニット欠損症の免疫学的研究

Fabp3 as a Biomarker of Skeletal Muscle Toxicity in the Rat: Comparison with Conventional Biomarkers

Fatty acid binding protein 3 (Fabp3) has been used as a serological biomarker of cardiac injury, but its utility as a preclinical biomarker of injury to skeletal muscle is not well described. Fabp3 concentrations were determined for tissues from Sprague-Dawley rats and found to occur at highest concentrations in cardiac muscle and in skeletal muscles containing an abundance of type I fibers, such as the soleus muscle. Soleus is also a primary site of skeletal muscle (SKM) injury caused by lipid-lowering peroxisome proliferator-activated receptor alpha (PPAR-alpha) agonists. In rats administered repeat doses of a PPAR-alpha agonist, the kinetics and amplitude of plasma concentrations of Fabp3 were consistent with plasma compound concentrations and histopathology findings of swollen, hyalinized, and fragmented muscle fibers with macrophage infiltration. Immunohistochemical detection of Fabp3 revealed focal depletion of Fabp3 protein from injured SKM fibers which is consistent with increased serum Fabp3 concentrations in treated rats. We then assessed the predictivity of serological Fabp3 for SKM necrosis in short duration toxicology studies. Rats were treated with various doses of 27 different compounds, and the predictivity of serological biomarkers was assessed relative to histology in individual rats and in treatment groups. Under these study conditions, Fabp3 was the most useful individual biomarker based on concordance, sensitivity, positive and negative predictive values, and false negative rate. In addition, the combination of Fabp3 and aspartate aminotransferase (AST) had greater diagnostic value than the conventional combination of creatine kinase-MM isoenzyme (CK) and AST.

Deficiencies of myocardial troponin-T and creatine kinase MB isoenzyme in dogs with idiopathic dilated cardiomyopathy

Abstract Objective To test the hypothesis that the derangement of myocardial energy homeostasis characteristic of idiopathic dilated cardiomyopathy (IDCM) of Doberman Pinschers was attributable to deficiency of creatine kinase (CK) isoenzyme MB relative to CK isoenzyme MM and troponin-T (Tn-T). Animals 9 Doberman Pinschers with advanced congestive heart failure attributable to IDCM and 9 mixed-breed dogs without cardiac disease (controls). Procedure Myocardial myofibrillar CK-MM, mitochondrial CK, and cytosolic CK-MB activities were determined in comparison with the concentration of cardiac-specific, myofibrillar protein Tn-T. Myocardial biopsy specimens were obtained after euthanasia and were ultrafrozen. Isoenzymes of CK were separated eloctrophoretically, and their activity was determined, using a coupled-enzyme, kinetic, fluorescent assay and densitometry. Troponin-T content was determined by immunoassay. Results Myofibrillar CK-MM and mitochondrial CK activities and Tn-T content were 25% lower in the dogs with IDCM than in controls, whereas cytosolic CK-MB activity was 50% lower. Conclusion Deficiency of CK-MB activity is a component of and may have a central role in the energy deficiency characteristic of failing myocardium in IDCM of Doberman Pinschers. A side-product of this study was the observation that, in dogs, CK-MB activity and Tn-T content of myocardium are sufficiently high to be candidate serum markers for active cardiac injury. (Am J Vet Res 1997;58:11–16)

Reversible dissociation and unfolding of dimeric creatine kinase isoenzyme MM in guanidine hydrochloride and urea.

The unfolding of dimeric cytoplasmic creatine kinase (MM) by guanidine hydrochloride and by urea has been investigated using activity measurements, far-ultraviolet circular dichroism, sedimentation velocity and fluorescence energy transfer experiments to monitor global structural changes. Intrinsic (cysteine and tryptophan residues) and extrinsic probes (1-anilinonaphthalene-8-sulfonate) were also used. The reversibility of the unfolding was checked by monitoring activity and tryptophan fluorescence. The unfolding of creatine kinase in guanidine hydrochloride is a reversible multistep process, as suggested by the non-coincidence of denaturation curves at equilibrium. Inactivation of the dimer precedes its dissociation into two monomers and an intermediate state was identified during the unfolding of the monomer. This intermediate state is characterized by a relatively high degree of secondary structure (as shown by far-ultraviolet circular dichroism), of compactness (as shown by fluorescence energy transfer measurements and sedimentation experiments), a fluctuating tertiary structure (as shown by near-ultraviolet circular dichroism) and a strong affinity for anilinonaphthalene sulfonate (as demonstrated by fluorescence). These results clearly indicate that the intermediate state detected possesses some of the properties of a molten globule. In urea, the unfolding pathway is reversible but differs from that observed in guanidine hydrochloride. Indeed inactivation, dissociation and loss of tertiary structure are coincident but the ellipticity curve is slightly shifted to a higher urea concentration. The dimer is dissociated into two expanded monomers possessing some secondary structure which is progressively lost at a higher urea concentration (6.4M). These results show that guanidine hydrochloride is approximately six times more effective than urea for inactivation and dissociation, underlining the fact that electrostatic interactions are very important in the stabilization of the active site and of the dimeric state.

Adenylate kinase mimics creatine kinase-MM isoenzyme in a CK isoenzyme electrophoresis assay.

Adenylate kinase activity (AK) originating from erythrocytes, present in hemolyzed serum behaves like creatine kinase MM isoenzyme (CK-MM) in some CK electrophoresis assays that employ, in their visualization reagent kits, adenosine monophosphate (AMP) as the sole inhibitor of AK, rather than a combination of AMP and a more potent inhibitor of erythrocyte AK, diadenosine pentaphosphate (Ap5A), to inhibit all contaminating-AK activities in serum and quantify only the CK isoenzyme activities in serum following electrophoretic fractionation on agarose gel. This can spuriously overestimate the CK-MM fraction and thereby result in underestimation of CK-MM or CK-BB isoenzymes if present. A hemolyzed serum sample obtained from an elderly patient was erroneously reported as containing low CK-MB due to such overestimation of CK-MM fraction in the sample. Supplementing the AMP already present in the visualization reagent formulation, used to estimate CK isoenzyme concentration in serum, with Ap5A can eliminate or effectively minimize AK interference, especially that caused by hemolysis, and thereby prevent reporting false-negative CK-MB result obtained with CK isoenzyme electrophoresis assays.

Measurement of creatine kinase isoforms by agarose gel electrophoresis in the diagnosis of myocardial infarction

To evaluate a method to quantitate the isoforms of serum creatine kinase isoenzyme MM (CK-MM) by agarose gel electrophoresis, sera of normal subjects (n = 74) and patients with acute myocardial infarction (n = 21) and other diseases (n = 67) were studied. The within-assay imprecision (CV) for CK-MM1, -MM2, and -MM3 was 1.9%, 0.8%, and 2.2% at the activity of 79, 105, and 64 U/L (30 degrees C, CK-NAC), respectively; while the assay-to-assay imprecision was 4.8%, 3.2% and 3.9%, respectively. The method could detect 5 U/L or more of any CK-MM isoform and was linear with respect to CK activity at values less than 1100 U/L. Sera from healthy subjects (n = 74) contained mainly CK-MM1 (mean = 48.5%), with lesser amounts of CK-MM2 (mean = 30.6%) and CK-MM3 (mean = 20.8%). The central 95-percentile reference range for the ratio of MM3/MM1 was 0.12-1.34 with mean = 0.49. The sensitivity of CK-MM3/MM1 ratio greater than 1.3 in the diagnosis of acute myocardial infarction employing the first available sample was 90% at a specificity of 91%, compared with a sensitivity of 81% and specificity of 87% for the conventional CK-MB assay. At CK-MM3/MM1 ratio of 1.6 or more, the specificity increased to 96% while sensitivity remained unchanged at 90%. This procedure for the quantitation of serum CK-MM isoforms is convenient, practical and suitable for inclusion in the routine panel of cardiac tests.

Heterogeneity of creatine kinase isoenzyme MM in serum in myocardial infarction: interconversion of the "normal" and "abnormal" sub-bands by glutathione

We used isoelectric focusing (IEF) in polyacrylamide gels to investigate the effects of glutathione on the sub-bands of serum creatine kinase (CK; EC 2.7.3.2) isoenzyme MM in acute myocardial infarction. The intensity of the "abnormal" sub-bands c (pI 7.25), e (pI 6.85), and g (pI 6.50) increased, and that of the "normal" sub-bands 1 (pI 6.91), 2 (pI 6.65), and 3 (pI 6.35) decreased, following serum incubation with reduced glutathione (GSH, final concentration 1.25 mmol/L). Further incubation with oxidized glutathione (GSSG, final concentration 5 mmol/L) reversed this change and restored the original pattern, whereas GSSG at 7.5 mmol/L caused sub-bands c, e, and g to disappear and sub-bands 1, 2, and 3 to be enhanced. Sequential incubation of serum with 2.5 mmol of GSSG and 7.5 mmol of GSH per liter produced the opposite sequence of events; i.e., the "abnormal" sub-bands disappeared then reappeared (and GSH at 10 mmol/L enhanced their reappearance). At higher concentrations, glutathione (GSH or GSSG) impaired the detection of the CK-MM sub-bands after IEF, an effect that was "quenched" by heat-inactivated serum of low CK activity. Likewise, the intensity of tissue CK-MM (corresponding to myocardium extracted into 100 mmol/L Tris HCl buffer, pH 7.4) was greatly enhanced by adding heat-inactivated serum to the tissue extract before IEF. We discuss the significance of these findings for the diagnosis of myocardial infarction.

Optimized preservation of isoforms of creatine kinase MM isoenzyme in plasma specimens and their rapid quantification by semi-automated chromatofocusing

We report a convenient chromatofocusing procedure for rapid and sensitive quantification of isoforms of the MM isoenzyme of creatine kinase (EC 2.7.3.2) in plasma and efficient methods for preserving isoform profiles during handling of specimens. The assay involves use of prepacked, re-usable Mono P chromatofocusing columns and a "Fast Protein Liquid Chromatograph" (FPLC) system with on-line detection of isoform enzymatic activity in column effluent. Profiles of isoforms are analyzed within 25 min with the use of a 1-mL column; the lower limit of sensitivity for CK activity is 5 mU, and recovery of each isoform is within 1% of the amount added to plasma. Collection of blood specimens in Vacutainer Tubes containing 28.5 mumol of EDTA (final concentration in plasma, 7 to 10 mmol/L) inhibited carboxypeptidase activity in plasma by 76%, sufficient to essentially abolish isoform conversion in vitro at room temperature. These methods should facilitate applications of isoform analysis for diagnosis of myocardial infarction and coronary artery recanalization.

Heterogeneity of serum creatine kinase isoenzyme MM in myocardial infarction: clinical significance and post-synthetic conversion of "abnormal" sub-bands.

We investigated the "abnormal" sub-bands of serum creatine kinase (CK; EC 2.7.3.2) isoenzyme MM in acute myocardial infarction (AMI), using isoelectric focusing in polyacrylamide gels, and compared the patterns with those for non-AMI patients and for athletes with increased CK. The "abnormal" sub-bands a (pI 7.55), b (pI 7.35), c (pI 7.25), d (pI 7.05), e (pI 6.85), f (pI 6.72), g (pI 6.50), h (pI 6.40), i (pI 6.28), j (pI 6.20), and k (pI 6.15), in common with "normal" sub-bands 1 (pI 6.91), 2 (pI 6.65), and 3 (pI 6.35), were less uniformly distributed in the AMI and demonstrated a faster rate of anodal conversion than in the non-AMI patients and athletes. "Abnormal" sub-bands a and b were detected only in the AMI patients. Incubation of myocardial CK-MM, predominantly CK-MM 1, with 2-mercaptoethanol, 15 mmol/L, resulted in conversion to the "abnormal" sub-bands b and c. Incubation of myocardial CK-MM with normal serum of low total CK yielded CK-MM 3, which on further incubation with 2-mercaptoethanol, 15 mmol/L, resulted in conversion to the "abnormal" sub-bands f and g. Comparable in vitro incubation of serum from AMI patients gave pattern changes consistent with conversion of CK-MM 1 to b, c; CK-MM 2 to d, e; and CK-MM 3 to f, g.

Prompt detection of myocardial injury by assay of creatine kinase isoforms in initial plasma samples.

To determine the sensitivity and specificity of assays of isoforms of the MM isoenzyme of creatine kinase (CK) and MB-CK for the diagnosis or exclusion of acute myocardial infarction in plasma samples obtained at the time of presentation to the emergency room of patients with suspected myocardial infarction, 50 patients in whom the diagnosis was ultimately established and 31 in whom infarction was ultimately excluded were evaluated. Of the 50 patients in whom infarction was ultimately established, 47 had an increased ratio of the MM3 to the MM1 isoform or elevated MB-CK in the plasma sample initially available. The specificity of the approach was 77%, sensitivity 94%. The availability of rapid assays for MM-CK isoforms and for MB-CK should facilitate determination of the presence or absence of acute myocardial injury sufficiently promptly so that decisions regarding treatment can be made optimally.

Detection of reperfusion within 1 hour after coronary recanalisation by analysis of isoforms of the MM creatine kinase isoenzyme in plasma

Early non-invasive detection of recanalisation within 1 hour of its implementation pharmacologically is needed to facilitate optimal selection of patients requiring further aggressive management of acute myocardial infarction. Recent results from studies of experimental animals suggest that prompt detection of reperfusion is possible based on analysis of sequential changes in the relative activities of individual isoforms of the MM isoenzyme of creatine kinase (CK) in plasma. To determine whether analogous changes occur in patients and whether they distinguish patients with from those without reperfusion, we evaluated CK isoform activities serially in plasma samples from 18 patients with reperfusion induced pharmacologically or by angioplasty and compared them with changes seen in 12 patients with infarction without reperfusion. In 11 of the 12 patients without reperfusion, the percentage in plasma of the MM CK isoform released from the heart, MM, did not change or decreased in the first hour. In contrast, among 17 of 18 patients with reperfusion, the percentage of MM, increased markedly over the first hour from a mean of 38 ± 2% to 53 ± 4%. Thus, 94% of patients ultimately shown to exhibit and 92% of those ultimately shown to fail to exhibit reperfusion were distinguished correctly. Accordingly, assessment of activities of MM CK isoforms in plasma should prove useful in differentiating patients with and without reperfusion at a time when additional interventions needed to consolidate the gains accompanied by initial pharmacological treatment can still be initiated effectively.

Stability and electrophoretic characteristics of creatine kinase BB extracted from human brain and intestine.

Creatine kinase (CK; EC 2.7.3.2) isoenzyme BB extracted from brains of rats reportedly undergoes modification at 37 degrees C, leaving an electrophoretic variant that accounts for most of the residual CK activity. This variant, called CK-BB', migrates on electrophoresis similarly to creatine kinase isoenzyme MB. Using electrophoresis and immunoinhibition with antiserum to creatine kinase isoenzyme MM, we found CK-BB to be the only identifiable cytoplasmic isoenzyme in surgical samples from human brain and intestine. In contrast, we found that some samples of brain obtained at autopsy contain CK-BB'. We also found that CK-BB extracted from human brain was converted to CK-BB' upon incubation in serum or plasma at 37 degrees C. We found a similar development of CK-BB' in incubation mixtures of serum or plasma containing CK-BB obtained from surgical samples of human intestine. The development of CK-BB' during infarction of the gastrointestinal system may thus be a source of false-positive CK-MB in the laboratory verification of myocardial infarction when electrophoresis is used as the only method to identify CK isoenzymes.

Identification of the carboxypeptidase responsible for the post-synthetic modification of creatine kinase in human serum

The enzyme responsible for the post-translational modification of creatine kinase-MM isoenzyme was purified from human plasma. The enzymatic activity of this enzyme (modifying protein) on the synthetic substrates hippuryl- l-arginine, hippuryl- l-lysine, 3-(2-furylacryloyl)- l-arginine and 3-(2-furylacryloyl)- l-alanyl- l-lysine and the ratio of activities on these substrates are in good agreement with the enzymatic activity of the human serum carboxypeptidase N. The effect of metal ions, chelating agents, proteolytic inhibitors and carboxypeptidase N inhibitor could not differentiate the modifying protein from human serum carboxypeptidase N. Affinity chromatography on Concanavalin-A-Sepharose demonstrated the glycoprotein nature of the modifying protein. The difference in molecular weight observed between modifying protein and carboxypeptidase N can be explained by known instability characteristics and the influence of proteolytic enzymes during purification. Double immunodiffusion analysis with purified antiserum to human carboxypeptidase N confirmed the identity of the modifying protein and carboxypeptidase N.

Heterogeneity of serum creatine kinase isoenzyme MM in myocardial infarction: standardization of patterns by use of cord-blood serum.

We investigated serum creatine kinase (CK; EC 2.7.3.2) isoenzyme MM in myocardial infarction, using isoelectric focusing in polyacrylamide gels. As many as 14 sub-band species were detected, and sequential sampling revealed a progressive anodal shift in their distribution. A nomenclature for sub-band typing is proposed. It denotes the major sub-bands as 1 (pl 6.91), 2 (pl 6.65), and 3 (pl 6.35); which are usually detected in sera of normal CK range. Abnormal sub-bands (associated with increases in the concentrations of CK) are denoted as a (pl 7.55), b (pl 7.35), c (pl 7.25), d (pl 7.05), e (pl 6.85), f (pl 6.72), g (pl 6.50), h (pl 6.40), i (pl 6.28), j (pl 6.20), and k (pl 6.15). Cord-blood sera gave highly reproducible CK-MM patterns characterized by prominent detection of sub-bands 1-3 and faint detection of c, e, f, g, h, i, and j. It is recommended as a standard for CK-MM sub-band typing.

Quantitative analysis for isoforms of creatine kinase MM in plasma by chromatofocusing, with on-line monitoring of enzyme activity.

Changes in the proportions of individual isoforms of the MM isoenzyme of creatine kinase (CK; EC 2.7.3.2) in plasma promptly reflect both myocardial infarction and coronary recanalization. However, quantitative methods developed thus far are too slow or cumbersome for routine use in making clinical decisions. We report a convenient, quantitative chromatofocusing assay with on-line fluorometric detection of isoform activity in the column eluent that provides results within 40 min from the time of sample application. Sample eluted from a microbore chromatofocusing column (1.8-mL bed volume) is split between a reaction stream, into which CK reagents are added, and a reference stream. After incubation at 37 degrees C, NADPH formed by reaction of isoforms with CK reagent is detected at 340 nm. The system can detect activity of individual isoforms in plasma samples having total CK activity greater than or equal to 21 U/L (30 degrees C). Results correlated closely with those obtained by previously validated, but slow, chromatofocusing (r = 0.98, n = 30) and protein immunoblotting (r = 0.90, n = 20) procedures.

Physiologic modelling of MM creatine kinase isoforms

We assessed the kinetics in vivo of isoforms of the MM isoenzyme of creatine kinase. A linear model accounting for plasma conversion of tissue isoform to two other isoforms and disappearance of each from the circulation was tested by non-linear least squares approximations to time-activity profiles of isoforms injected intravenously in dogs. The model was validated by analysis of disappearance rate constants after injections of isoforms with plasma conversion inhibited. The data show that disappearance is the primary route of egress of isoform activity from the circulation and the tissue isoform has the fastest disappearance rate.

Time dependent conversion of creatine kinase MM isoforms in man.

Subforms of creatine kinase MM isoenzyme (isoforms, pI: MMA = 7.95, MMB = 7.76, MMC = 7.54) in human myocardium and serum were quantified by chromatofocusing. Creatine kinase MMA was a dominant isoform (greater than 94% of MM) in normal (n = 3) and in both infarcted and non-infarcted myocardium (n = 2). To investigate isoform conversion in vitro partially purified MMA was incubated with human plasma at 37 degrees C for 24 h (n = 5). Creatine kinase MMB (0%, 47%, 44% of MM at 0, 12, 24 h respectively) and MMC (0%, 22%, 42%) sequentially appeared in incubation media whereas MMA (100%, 31%, 14%) disappeared rapidly with a mean disappearance rate of -0.00169(0.00021)(SD) min-1. Individual differences in conversion velocity were small (SD less than 5%). To investigate isoform conversion in vivo serum isoforms were analysed in patients with acute myocardial infarction (n = 7). MMA was first dominant (A:B:C = 54:34:12%) in the early stage (6-9 h after the onset of chest pain) followed by MMB dominant (19:42:39%) in the middle stage (24-35 h), and MMC dominant (6:22:72%) in the late stage (54-60 h). Changes in isoform proportion were time dependent regardless of serum creatine kinase activity. These findings are consistent with canine isoform conversion reported previously except that in man the velocity of conversion was slower than in the dog. Thus analysis of serum creatine kinase MM isoforms may allow the onset of acute myocardial infarction to be precisely dated. Moreover, determination of MMA, the isoform native to myocardium with a short serum half life, may be useful in the prompt diagnosis of myocardial infarction.

Quantification of isoforms of plasma MM creatine kinase (CK) with an immunoblot procedure.

The MM isoenzyme of creatine kinase, a dimer composed of two M ("muscle type") subunits, is found in myocardium, where it constitutes 85% of tissue CK, and in skeletal muscle, where it constitutes virtually 100%, as well as in other tissues. The tissue form is designated MMA. When MMA circulates in plasma, it undergoes stepwise, post-translational modification, mediated by proteolytic enzymes in plasma and giving rise to isoforms called MMB and MMC, which lack carboxy terminal lysine on one or two subunits, respectively. We have shown previously that changes with time in plasma profiles of MM creatine kinase (CK) isoforms in dogs reflect myocardial infarction within 1 hour after the onset of coronary occlusion and permit noninvasive detection of reperfusion within 30 minutes after release of an occlusive coronary arterial ligature. However, analysis of MM CK isoforms in plasma from patients has been hampered by the lack of availability of quantitative as opposed to qualitative methods. This study was performed to develop and validate an assay with the sensitivity and specificity needed for accurate quantification of MM CK isoforms in samples of plasma from patients. A rapid assay procedure will be required ultimately for prospective, clinical use. However, as a first step and for use in development and standardization of rapid assays, a procedure is needed for accurate qualification of isoforms even if its implementation is laborious and slow. The isoform composition of normal plasma was found to comprise 32.0% MMA, 34.9% MMB, and 32.7% MMC.(ABSTRACT TRUNCATED AT 250 WORDS)