Transamidase site-targeted agents alter the conformation of the transglutaminase cancer stem cell survival protein to reduce GTP binding activity and cancer stem cell survival.

Type 2 transglutaminase (TG2) is an important cancer stem cell survival protein that exists in open and closed conformations. The major intracellular form is the closed conformation that functions as a GTP-binding GTPase and is required for cancer stem cell survival. However, at a finite rate, TG2 transitions to an open conformation that exposes the transamidase catalytic site involved in protein-protein crosslinking. The activities are mutually exclusive, as the closed conformation has GTP binding/GTPase activity, and the open conformation transamidase activity. We recently showed that GTP binding, but not transamidase activity, is required for TG2-dependent cancer stem cell invasion, migration and tumour formation. However, we were surprised that transamidase site-specific inhibitors reduce cancer stem cell survival. We now show that compounds NC9, VA4 and VA5, which react exclusively at the TG2 transamidase site, inhibit both transamidase and GTP-binding activities. Transamidase activity is inhibited by direct inhibitor binding at the transamidase site, and GTP binding is blocked because inhibitor interaction at the transamidase site locks the protein in the extended/open conformation to disorganize/inactivate the GTP binding/GTPase site. These findings suggest that transamidase site-specific inhibitors can inhibit GTP binding/signalling by driving a conformation change that disorganizes the TG2 GTP binding to reduce TG2-dependent signalling, and that drugs designed to target this site may be potent anti-cancer agents.

Effect of caffeine on target detection and rifle marksmanship.

Thirteen healthy and rifle-trained male military reservists performed shooting sessions on two separate occasions 1 h following the ingestion of placebo or 300 mg of caffeine. Shooting included both friend-foe (FF) and vigilance (VIG) tasks, and were performed in the following order: two FF sequences (4 min each), four VIG sequences (30 min each), and two additional FF sequences. The shooting sessions lasted approximately 2.5 h under outdoor conditions (air temperature range from - 3 to 14 degrees C) and were held 48 h apart in a counter-balanced order. Performance measures during the shooting session included engagement time, friend-foe discrimination, and marksmanship accuracy and precision. Assessments of thermal comfort, tiredness, and debilitating symptoms preceded and followed the shooting session, while a self-assessment on performance was administered post-shooting only. Blood was sampled immediately prior to the beginning of the shooting session and was used to determine plasma caffeine, cortisol, and testosterone levels. Engagement times were faster and certain measures of accuracy and precision were impaired during the later FF and VIG sequences. However, caffeine ingestion had no affect upon any of the marksmanship measures, although it did alleviate cold stress and tiredness. That caffeine ingestion did not affect target detection and rifle marksmanship is a finding that differs from other studies, and is explained by a beneficial arousal caused by the mild level of cold stress experienced by the participants.

Synthesis and evaluation of novel dipeptide-bound 1,2,4-thiadiazoles as irreversible inhibitors of guinea pig liver transglutaminase.

Herein we report the synthesis and evaluation of 14 novel peptides as potential irreversible inactivators of guinea pig liver transglutaminase (TGase). These peptides were designed to resemble Cbz-L-Gln-Gly, known to be a good TGase substrate, and to include a 1,2,4-thiadiazole group. The side chain length of the amino acid residue bearing the inhibitor group was also varied in order to permit investigation of this effect. Their inactivation rate constants were measured using a direct continuous spectrophotometric method and were found to vary between 0.330 to 0.89 microM(-1) min(-1).

Nonlinear free energy relationship in the general-acid-catalyzed acylation of rat kidney gamma-glutamyl transpeptidase by a series of gamma-glutamyl anilide substrate analogues.

The gamma-glutamyl transpeptidase (GGT) purified from rat kidney reacts with a series of eight parasubstituted L-glutamyl gamma-anilides, in the presence of Gly-Gly, catalyzing the formation of gamma-Glu-Gly-Gly (pH 8.0, 37 degrees C). The transpeptidation reaction was followed through the discontinuous colorimetric determination of the concentration of released parasubstituted aniline. Steady-state kinetic studies were performed to measure k(cat) and K(M) values for each anilide substrate. A Hammett plot constructed by the correlation of log(k(cat)) and the sigma(-) parameter for each anilide substrate displays statistically significant upward curvature, consistent with a general-acid-catalyzed acylation mechanism in which the geometry of the transition state changes with the nature of the para substituent. Kinetic isotope effects were measured and are consistent with a reaction involving a proton in flight at the rate-limiting transition state. The pH-rate profiles measured over pH 7.0-9.5 are bell-shaped with kinetic pK(a) values that may be attributed to the active site nucleophile (or its general-base catalytic partner) and the active-site general acid. The variation of the latter pK(a) value as a function of temperature is consistent with an enthalpy of ionization expected for an ammonium ion acting as a general acid. Examination of the variation of k(cat) as a function of temperature gave values for the enthalpy and entropy of activation that are similar to those determined for the general-acid-catalyzed breakdown of the tetrahedral intermediate formed during acylation of chymotrypsin by similar amide substrates.

Kinetic studies of guinea pig liver transglutaminase reveal a general-base-catalyzed deacylation mechanism.

Guinea pig liver transglutaminase (TGase) reacts with 0.1 mM N-Cbz-L-Glu(gamma-p-nitrophenyl ester)Gly (5, prepared herein, K(M) = 0.02 mM) to undergo rapid acylation that can be followed spectrophotometrically at 400 nm (pH 7.0, 25 degrees C). Deacylation of the transiently formed thiolester acyl enzyme intermediate via catalytic aminolysis was studied in the presence of six primary amines of widely varying basicity (pK(NH+) = 5.6-10.5). Steady-state kinetic studies were performed to measure k(cat) and K(M) values for each amine substrate. A Brønsted plot constructed through the correlation of log(k(cat)/K(M)) and pK(NH+) for each amine substrate displays a linear free-energy relationship with a slope beta(nuc) = -0.37 +/- 0.08. The shallow negative slope is consistent with a general-base-catalyzed deacylation mechanism in which a proton is removed from the amine substrate during its rate-limiting nucleophilic attack on the thiolester carbonyl. Kinetic isotope effects were measured for four acceptor substrates (water, kie = 1.1 +/- 0.1; aminoacetonitrile, kie = 5.9 +/- 1.2; glycine methyl ester, kie = 3.4 +/- 0.7; N-Ac-L-lysine methyl ester, kie = 1.1 +/- 0.1) and are consistent with a proton in flight at the rate-limiting transition state. The active site general-base implicated by these kinetic results is believed to be His-334, of the highly conserved TGase Cys-His-Asp catalytic triad.

Evaluation of novel dipeptide-bound alpha,beta-unsaturated amides and epoxides as irreversible inhibitors of guinea pig liver transglutaminase.

Herein, we report the results of irreversible inhibition of guinea pig liver transglutaminase (TGase) by a series of 24 novel dipeptides containing either an alpha,beta-unsaturated amide or an epoxide functional group. Their inactivation rate constants were measured using a direct continuous spectrophotometric method and were found to vary between 421 x 10(3) and 3000 x 10(3)M(-1)min(-1).

A direct continuous spectrophotometric assay for transglutaminase activity.

Herein we report the development of a direct and continuous spectrophotometric method for determining transglutaminase (TGase) activity by using N,N-dimethyl-1,4-phenylenediamine (DMPDA) as a gamma-glutamyl acceptor substrate and carbobenzyloxy-l-glutamylglycine (Z-Gln-Gly) as a typical peptide gamma-glutamyl donor substrate. The transamidation activity of TGase can thus be followed by monitoring the increase of absorbance of the resulting anilide product at 278 nm. The extinction coefficient of the authentic, independently synthesized anilide was determined to be epsilon = 8940 +/- 55 M(-1) cm(-1). Using this assay, we determined the apparent K(M) of DMPDA to be 0.25 mM, which compares favorably to the apparent K(M) values determined for other acceptor substrates under conditions where Z-Gln-Gly is also used as the donor substrate, such as N-acetyl-l-lysine methyl ester (9.6 mM) and methylamine (13.1 mM). Finally, the sensitivity of this assay technique was established through the measurement of irreversible inhibition constants for iodoacetamide, determined to be K(I) = 75 +/- 11 nM and k(inact) = (120 +/- 1) x 10(5) M(-1) min(-1).

Increased discrimination of "false memories" in autism spectrum disorder.

Individuals with autism spectrum disorder (ASD) have impaired ability to use context, which may manifest as alterations of relatedness within the semantic network. However, impairment in context use may be more difficult to detect in high-functioning adults with ASD. To test context use in this population, we examined the influence of context on memory by using the "false memory" test. In the false memory task, lists of words were presented to high-functioning subjects with ASD and matched controls. Each list consists of words highly related to an index word not on the list. Subjects are then given a recognition test. Positive responses to the index words represent false memories. We found that individuals with ASD are able to discriminate false memory items from true items significantly better than are control subjects. Memory in patients with ASD may be more accurate than in normal individuals under certain conditions. These results also suggest that semantic representations comprise a less distributed network in high-functioning adults with ASD. Furthermore, these results may be related to the unusually high memory capacities found in some individuals with ASD. Research directed at defining the range of tasks performed superiorly by high-functioning individuals with ASD will be important for optimal vocational rehabilitation.

Guinea pig liver transglutaminase: A modified purification procedure affording enzyme with superior activity in greater yield.

Tissue transglutaminase purified from guinea pig livers has a very broad substrate specificity in comparison with other members of the transglutaminase family and therefore is useful for substrate analogue kinetic studies. Modifications made in our laboratory to the standard purification protocol (J. E. Folk and S. I. Chung, 1985, Methods Enzymol. 113, 358-364) have yielded a 28% increase in specific activity and 55% increase in overall yield, while reducing the number of steps to the purification. Herein we report some of the highest yields and specific activities for guinea pig liver transglutaminase found in the literature, as well as the use of lyophilization as a solution to the long-standing problem of enzyme stability during storage.

Synthesis and characterization of a series of novel glutamic gamma-15N-anilide dipeptides.

The preparation of a series of novel Cbz-Gln-Gly dipeptide derivatives is reported, wherein the gamma-carboxamide groups of the glutamine side chains have been modified to gamma-15N-anilides which are substituted in the para position with -NO2, -Cl, -H, -CH3, -OCH3, and -N(CH3)2. Characterization of the free anilines (p(kappa)a values and 15N NMR chemical shifts) and corresponding gamma-anilides (15N NMR chemical shifts and FTIR wavenumbers) is also reported. Correlation of these physicochemical data to Hammett substituent parameters ((sigma)para) is discussed. These novel dipeptide derivatives should prove to be generally useful for structure-function enzymology studies of gamma-glutamyl transferring enzymes.

Phosphorylation of the sodium--potassium adenosinetriphosphatase proceeds through a rate-limiting conformational change followed by rapid phosphoryl transfer.

The sodium-potassium adenosinetriphosphatase of sheep kidney, preincubated with sodium and magnesium (E.Nae), reacts with 0.01-2.00 mM ATP to form covalent phosphoenzyme (E-P). The first order rate constant for phosphorylation increases hyperbolically with ATP concentration with a maximum value of (4.6 +/- 0.9) x 10(2) s-1 and K0.5 = 75 +/- 25 microM (ph 7.4, 25 degrees C, 120 mM NaCl, and 3 mM MgCl2). If the phosphoryl-transfer step were rate-limiting, the approach to equilibrium to give 50% E-P in the presence of ADP would follow kobsd=Kf+Kr+9.2 x 10(2) s-1. However, the formation of phosphoenzyme from E.Na3 with 1.0 mM ATP plus 2.0 mM ADP proceeds to 50% completion with kobsd=(4.2 +/- 0.8) x 10(2) s-1. This result show that phosphoryl transfer from bound ATP to the enzyme is not the rate limiting step for phosphoenzyme formation from E.Na3. The result is consistent with a rate-limiting conformational change of the E.Na3.ATP intermediate that is followed by rapid phosphoryl transfer, with kcat > or = 3000 s-1.

Differences in the visual control of pantomimed and natural grasping movements.

In a series of experiments, we studied the differences between natural target-directed grasping movements and 'pantomimed' movements directed towards remembered objects. Although subjects continued to scale their hand opening for object size when pantomiming, grip formation and other kinematic variables differed significantly from those seen in normal target-directed actions. This was true whether the subjects had just seen the target object 2 sec before (Experiments 1 and 2) or whether the target object was still present and they were simply required to pantomime the grasping movement beside it (Experiment 3). We argued that these pantomimed reaches were being driven by stored perceptual information about the object, and were not utilizing the normal visuomotor control systems that direct actions in real time. This interpretation received strong support from observations of a patient with visual form agnosia who was also tested. In an earlier report, we had shown that this patient showed anticipatory scaling of her grasp despite her inability to discriminate between objects perceptually on the basis of size. The present study showed, however, that the requirement to remember an object even briefly, or to pantomime an action beside it, was enough to completely disrupt her visuomotor scaling (Experiments 2 and 3). That this reflected a failure of perception rather than imagery or understanding was supported by the fact that she could convincingly pantomime actions to imagined, familiar objects, the sizes of which were known to her (Experiment 4). All these results suggest that the mechanisms underlying the formation of perceptual representations of objects are quite independent of those mediating on-line visuomotor control.

Immunohistologic study of T-cell receptor delta-chain expression in rheumatoid synovial membranes.

Lymphocytes expressing gamma delta T-cell receptors (TCRs) have been shown to be reactive to mycobacterial antigens as well as the so-called stress proteins. The detection of increased numbers of gamma delta cells in the synovial fluid and peripheral blood of some patients with rheumatoid arthritis has suggested a potential role for these lymphocytes in the pathogenesis of this disorder. Twenty-three rheumatoid synovial membranes were studied using immunohistology and monoclonal antibodies in an attempt to define the patterns of distribution of gamma delta T cells in rheumatoid synovitis. Consecutive sections were stained for T1(CD5), T4(CD4), T8(CD8), TAC(CD25), the delta-chain markers delta TCR1 and delta TCS1, and the beta-chain marker beta F1. Our results show some regional differences in the distribution of CD4 and CD8 cells, the former being prominent in the lymphocytic aggregates and the latter most prominent in diffuse infiltrates immediately adjacent to the synovial lining layer. All tissues showed extensive staining for beta F1; an estimated average of more than 90% of T cells expressed alpha beta TCR. The majority of samples showed limited staining for both delta-chain antibodies, with 20 of the 23 tissues appearing to have less than 1% of T lymphocytes expressing these markers. Three tissues stained extensively for both delta TCR1 and delta TCS1 in particular areas of the section. In these areas, small perivascular lymphocytic aggregates appeared to be composed mainly of gamma delta cells. TAC staining was virtually absent in all areas and tissues. It was concluded that the majority of T lymphocytes infiltrating rheumatoid synovial membranes express alpha beta TCR.(ABSTRACT TRUNCATED AT 250 WORDS)

The response of poorly differentiated prostatic tumors to staining for prostate specific antigen and prostatic acid phosphatase: a comparative study.

A comparative study was done of the usefulness of immunohistochemical stains for prostate specific antigen and prostatic acid phosphatase in 20 poorly differentiated prostatic tumors. The stain for prostatic acid phosphatase was preferable to the prostate specific antigen stain not only because it was more intense and, therefore, more visible but also because it often was positive in areas in which an equivocal or negative stain was obtained with prostate specific antigen.