Development and validation of a questionnaire measuring quality of life in primary caregivers of children with atopic dermatitis (QPCAD).

BACKGROUND: Disease-specific health-related quality of life (HRQoL) instruments for primary caregivers of children with atopic dermatitis are useful in evaluating the efficacy of treatment in clinical practice and study. However, no such scale has been available in Japan. OBJECTIVES: To develop and validate a self-administered instrument specifically designed to measure quality of life in primary caregivers of children with atopic dermatitis (QPCAD). METHODS: This study consisted of three successive phases: the item generation phase, pilot test phase and validation phase. In the item generation phase, questionnaire items were derived from 33 qualitative interviews with primary caregivers. In the pilot test phase, the face and content validity of the preliminary scale were assessed (n = 33). In the validation phase, the questionnaire was finalized and assessed in terms of statistical performance (n = 416). RESULTS: The QPCAD included 19 items in the following categories: 'exhaustion', 'worry about atopic dermatitis', 'family cooperation' and 'achievement'. The reliability of internal consistency was fair (Cronbach's alpha coefficients 0.66-0.87). The QPCAD subscales and total score were significantly correlated with psychological health, physical health, anxiety, depression and severity score, with mild to moderate correlation coefficients. Test-retest reliability and responsiveness to change in severity were also satisfactory. CONCLUSIONS: The QPCAD is an appropriate tool for assessing HRQoL of primary caregivers of children with atopic dermatitis in clinical practice and clinical trials.

Inhibition of human DNA polymerase alpha by alpha 1-antichymotrypsin.

alpha 1-Antichymotrypsin (ACT), which is known as an efficient serum protease inhibitor and is detected in tumor cell nuclei, was found to inhibit the activity of DNA polymerase alpha purified from human stomach adenocarcinoma. The concentration of ACT required for 50% inhibition was 1.0 mg/ml and the manner of its inhibition showed the partially competitive relationship between ACT and DNA in the assay system. Furthermore the removal of ACT by anti-ACT antibody lost its antichymotryptic and anti-DNA polymerase activities in parallel. On the other hand, it did not inhibit the activity of human DNA polymerase beta. Other human serum proteins including serum albumin, alpha 1-acid glycoprotein, alpha 1-antitrypsin, and immunoglobulin G as well as other protease inhibitors such as leupeptin, pepstatin, phenylmethylsulfonyl fluoride, and chymostatin did not affect the activity of DNA polymerase alpha. Furthermore ACT heated at 60 degrees C did not inhibit DNA polymerase alpha, although it could still bind to DNA as well as native ACT. It was therefore concluded that the inhibitory action of ACT on DNA polymerase alpha was a direct phenomenon unrelated to its protease inhibitory or DNA binding activities.

Incorporation of alpha-1-antichymotrypsin into carcinoma cell nuclei of human stomach adenocarcinoma transplanted into nude mice.

Human stomach adenocarcinomas containing alpha-1-antichymotrypsin (ACT) in their cell nuclei were transplanted into nude mice. The presence of ACT was monitored using an immunohistochemical technique with horseradish peroxidase-labeled rabbit anti-ACT Fab' as well as single radial immunodiffusion. Two weeks after transplantation, ACT could be found neither in transplanted carcinoma cells nor in the sera of carcinoma-bearing nude mice. However, if human ACT was injected i.v., it could be detected in the transplanted carcinoma cell nuclei 2 h after injection. The ACT was detected immunohistochemically and was confirmed by biochemical fractionation using 125I-labeled ACT. On the other hand, the amount of ACT production was not sufficient to indicate biosynthesis. These results demonstrated that ACT detected in stomach carcinoma cell nuclei was not synthesized in carcinoma cells but was incorporated from the blood circulation.

High molecular mass type i.v. collagen-specific metalloprotease from human carcinoma tissue.

A protease degrading type IV collagen was purified more than 8000-fold from human stomach carcinoma tissue. This protease degraded type IV collagen, while type I, II, III and V collagen, laminin, fibronectin, casein, albumin and hemoglobin were not affected. This enzyme had a pH optimum of pH 7.0-8.0 and was inhibited completely by EDTA and o-phenanthroline, but not by seryl, thiol and carboxyl protease inhibitors. Furthermore, the molecular mass of this enzyme was estimated to be 1 MDa by Sepharose 6B column and HPLC-gel filtration. The molecular mass and substrate specificity of this metalloprotease from human carcinoma tissue indicate it to be a new protease.

Effect of dietary proteins on the turnover of rat liver argininosuccinate synthetase.

The rates of synthesis and degradation of arginosuccinate synthetase in rat liver under various dietary conditions were determined. The relative rate of the enzyme synthesis in the livers of rats fed on 70% casein diet was 4.0 times greater than that for rats fed on 5% casein diet. The rate constants of degradation (Kd of argininosuccinate synthetase were estimated to be 0.15 and 0.16 day-1 under 70% and 5% casein feeding, respectively. When the dietary conditions were changed acutely from 70% to 5% casein diet or vice versa, the rates of the enzyme synthesis decreased or increased, respectively, and the rates of enzyme degradation were also affected. The change from 5% to 70% casein diet caused a transient decrease in the rate of degradation. After the enzyme activity had achieved a new steady-state level, the enzyme degradation proceeded at the normal steady rate. On the other hand, the change from 70% to 5% casein diet caused a transient increase in the rate of degradation. Thus, the only factor regulating the amount of enzyme in rat liver is the rate of enzyme synthesis under the steady-state conditions. However, the rates of both enzyme synthesis and degradation are involved in the regulation of the amount of enzyme during dietary transition.

Studies on rat liver argininosuccinate synthetase. Inhibition by various amino acids.

Inhibition studies of crystallized rat liver argininosuccinate synthetase [EC 6.3.4.5] are described. 1. L-Argininosuccinate, L-histidine, and L-tryptophan inhibited the enzyme activity at saturating amounts of the substrates. 2. L-Norvaline, L-argininosuccinate, L-arginine, L-isoleucine, and L-valine competitively inhibited the enzyme activity at a low concentration of L-citrulline, with Ki values of 1.3 x 10(4) M, 2.5 X 10(-4) M, 6.7 X 10(-4) M, 6.3 X 10(-4) M, and 6.0 x 10(-4) M, respectively. 3. L-Argininosuccinate and L-arginine competitively inhibited the enzyme activity at a low concentration of L-aspartate, with Ki values of 9.5 x 10(-4) M and 1.2 x 10(-3) M, respectively. 4. The modes of inhibition by L-histidine were mixed-noncompetitive, uncompetitive, and noncompetitive types with respect to L-citrulline, L-aspartate, and ATP, respectively. 5. When the enzyme was preincubated with L-citrulline, the enzyme activity was slightly increased in the presence of a low concentration of L-histidine in the assay mixture. 6. The conformation of the enzyme was markedly changed by the addition of L-histidine as judged from the CD spectrum. This change was partially reversed by incubation with L-citrulline.

The biological activity of alpha-1-antichymotrypsin: the change of chymotrypsin-inhibitory and immunoenhancing activities by heat treatment.

The relationship between chymotrypsin-inhibitory and immunoenhancing activity of alpha-1-antichymotrypsin was studied. alpha-1-Antichymotrypsin was treated at 50 degrees C, 55 degrees C or 60 degrees C for 15 min. It was found that antichymotryptic activity was reduced by half when alpha-1-antichymotrypsin was heated at 55 degrees C and was not detected at all when heating was carried out at 60 degrees C. alpha-1-Antichymotrypsin which was heated at 60 degrees C did not form a complex with chymotrypsin, but became a substrate for chymotrypsin. The effect of native and heated alpha-1-antichymotrypsin on antibody response was studied in mice. alpha-1-Antichymotrypsin increased the number of anti-sheep erythrocytes antibody producing cells even when it was heated at 60 degrees C. Circular dichroism and single radial immunodiffusion were used to detect conformational changes. Circular dichroism in the region of side chain absorption showed that the intensities of the spectra at 296, 284, and 265 nm decreased with a rise in temperature from 50 to 60 degrees C. In single radial immunodiffusion analysis, alpha-1-antichymotrypsin did not form a halo after being heated at 60 degrees C. In conclusion, when alpha-1-antichymotrypsin was heated at 60 degrees C, the immunoenhancing activity remained intact while the antichymotryptic activity was lost with the conformational change.

Comparison of the urea cycle in conventional and germ-free mice.

The urea cycle in the liver of conventional mice was compared with that of germ-free mice. Among the urea cycle enzymes and urea cycle-related substances, only ornithine in the liver of the conventional mice was markedly more abundant than in that of the germ-free mice. The significance of this finding was supported by the comparison of the urea synthesis in the liver slices from these two types of mice.

Glucocorticoids reduce tachykinin NK2 receptor expression in bovine tracheal smooth muscle.

Neurokinin A is not only a potent bronchoconstrictor, but also has immuno-modulatory effects in animals and man, mediated via tachykinin NK2 receptors. We have examined the effect of the glucocorticoid, dexamethasone, on tachykinin NK2 receptor mRNA and the number of tachykinin NK2 receptors in bovine tracheal smooth muscle in vitro by Northern blot analysis using a human tachykinin NK2 receptor cDNA probe and receptor binding assay using [3H]SR48968 [(S)-N-methyl-N[4-acetylamino-4-phenylpiperidino-2(3,4-dichlorophenyl) butyl]benzamide]. Tachykinin NK2 receptor mRNA showed a time-dependent suppression (62% reduction after 6 h at 10(-7) M of dexamethasone), as well as a concentration-dependent suppression after the incubation with dexamethasone (IC50 = 1.3 x 10(-8) M). This suppression was abolished by the glucocorticoid receptor antagonist, mifepristone (RU38486), indicating that dexamethasone acts via the glucocorticoid receptor. It was also abolished by the protein synthesis inhibitor, cycloheximide (10 microg/ml), indicating that new protein synthesis is required on this suppression. Using the RNA polymerase inhibitor actinomycin D (5 microg/ml), we showed that the stability of tachykinin NK2 receptor mRNA was not affected by dexamethasone (t1/2 = 5 h). Nuclear run-on assays revealed a 51% reduction in the rate of tachykinin NK2 receptor gene transcription after treatment with dexamethasone for 6 h. Radioligand binding assay using an selective tachykinin NK2 receptor antagonist, [3H]SR48968 showed a significant decrease in the number of receptor binding sites after 16 h (Bmax = 262 +/- 23 versus 213 +/- 13 fmol/mg protein for vehicle and dexamethasone treatment respectively, P < 0.05), with no significant change at the earlier time points. These results suggest that glucocorticoids act on glucocorticoid receptors to decrease tachykinin NK2 receptor expression by decreasing the rate of tachykinin NK2 receptor gene transcription.

Argininosuccinate synthetase activity in cultured skin fibroblasts of citrullinemic patients.

The activities and kinetic properties of argininosuccinate synthetase in cultured skin fibroblasts of three citrullinemic patients with qualitative or quantitative abnormalities of hepatic argininosuccinate synthetase were examined. The cultured skin fibroblasts of a citrullinemic patient with a low hepatic argininosuccinate synthetase activity showed low activity and abnormal kinetics similar to those of the hepatic enzyme. This suggests a similar genetic origin of the fibroblast argininosuccinate synthetase as of the hepatic enzyme. However, we found normal argininosuccinate synthetase activity in cultured fibroblasts of two citrullinemic patients who had a low hepatic enzyme activity resulting from decrease in the amount of enzyme protein but with normal kinetic properties. This suggests that part of the quantitative abnormality of hepatic argininosuccinate synthetase of citrullinemic patients may be due to an abnormal organ-specific gene expression in the liver.

Qualitative and quantitative abnormalities of argininosuccinate synthetase in citrullinemia.

Enzymological and immunochemical analyses of the liver were preformed in seven Japanese patients with citrullinemia. Among the urea cycle enzymes in the liver, only the activity of argininosuccinate synthetase was specifically decreased to 2 to 50% of normal controls. Liver argininosuccinate synthetase of patients was indistinguishable from that of controls when tested immunochemically by Ouchterlony's double immunodiffusion technique with anti-rat argininosuccinate synthetase antiserum. Immunochemical analysis by means of the single radial immunodiffusion revealed that the decrease in the activity of liver argininosuccinate synthetase was explainable by a decrease in the amount of the enzyme protein in five patients, while the decrease in the activity in the other two patients was not accompanied by a decrease of enzyme protein. The Km values for the substrates of liver argininosuccinate synthetase of the former five were similar to those of the control, while the kinetic properties of the latter two were quite different in terms of higher Km values and negative cooperativity. From these results, we consider that citrullinemia may consist of more than one type including qualitative or quantitative abnormalities of argininosuccinate synthetase caused by some defects in certain genes or in the epigenetic processes in the liver.

Anorexigenic substance isolated from feces of rat and mouse.

A substance was isolated from the feces of conventional rats and mice which were fed laboratory diets. Marked reduction in food intake occurred for a few hours after intraperitoneal administration of this substance, while water intake also decreased. Two hr after the injection, when the anorectic effect appeared to be the strongest, no change was found in body temperature or blood glucose, but free amino acids in plasma were decreased. A comparative study using germfree and conventional mice indicated that the anorexigenic substance was produced by gastrointestinal microflora, since the yields of the anorexigenic substance from germfree mice was less than one tenth of that from conventional mice. A partially purified form of the substance, with large molecular weight, was isolated by Sephadex G-150 fractionation. It contained protein but the anorexigenic activity was not diminished by protein digestion.

Feeding suppression induced by a fecal anorexigenic substance (FS-T).

Intraperitoneal (IP) injection of a fecal anorexigenic substance (FS-T) induced significant suppression of feeding and this suppression recovered on the second day. At 2 hr after IP injection, at the time of maximum feeding suppression, plasma glucose, insulin and free fatty acid (FFA) levels did not change but amino acid level decreased. Intra-third cerebral ventricle (ICV) infusion of FS-T induced parallel but more potent feeding suppression. Analysis of meal patterns demonstrated that suppression of feeding after ICV treatment continued into the second day. FS-T was applied electrophoretically to glucose-sensitive and non glucose-sensitive neurons in the lateral hypothalamic area (LHA) and to glucoreceptor and non glucoreceptor neurons in the ventromedial hypothalamic nucleus (VMH). It significantly inhibited glucose-sensitive neurons but not non glucose-sensitive neurons, and excited both neuron types in the VMH. FS-T might thus work directly through the hypothalamic feeding control centers to suppress feeding. Even after pronase treatment of FS-T, a non-dialysable fraction of large molecular weight, consisting of protein and carbohydrate, maintained the original anorexigenic activity.

Objective monitoring of graft-versus-host disease: alpha 1-antichymotrypsin concentration changes after bone marrow transplantation in patients developing graft-versus-host reactions.

The levels of alpha 1-antichymotrypsin (ACT) was monitored in patients who underwent bone marrow transplantation. Seven received HLA-identical sibling bone marrow grafts, two received transplants from twins and one was given HLA-nonidentical marrow from his father. A dramatic increase of ACT was observed in all patients who developed graft-versus-host disease (GvHD). ACT did not rise at all in the case of patients who received marrow from twins, even in a patient who was given three transplants from the same donor. The patient transplanted from his father died from GvHD and the increase of ACT was the greatest fluctuation measured.

Alpha-1-antichymotrypsin (ACT) as an immune regulatory agent.

This study was carried out to elucidate the interaction of alpha-1-antichymotrypsin (ACT) with the immune reaction induced by interleukin-2 (IL-2). ACT, isolated from human sera and determined to be a single glycoprotein, was added to human peripheral T-cell cultured with IL-2. It was found that ACT effectively suppressed T-cell activation induced by IL-2, but the suppressive effect of ACT was inversely proportional to the concentration of IL-2. A similar effect was found when T-cells were stimulated with PHA. ACT had a suppressive effect when suboptimal concentrations of PHA were used, however this suppressive effect could not be found at the optimal PHA concentration. These findings suggest that ACT may be a regulatory agent in immune reactivity where low concentrations of IL-2 are present. Even under normal conditions, an individual is surrounded and invaded by numerous foreign elements and it may be that small amounts of IL-2 are constantly produced, which would indefinitely trigger chained immune reactions if there were no regulatory agent like ACT.

Binding of modified alpha-1-antichymotrypsin to mitogen-stimulated human lymphocyte membrane: a model for immune suppression.

Alpha-1-antichymotrypsin (ACT), an acute phase reactant protein elevated during acute inflammation, and its derivatives (asialo ACT and acid-exposed asialo ACT) were investigated their effect on lymphocyte proliferative responses, and evidence for binding to lymphocyte membranes as well as the characteristics of this binding were investigated. Acid-exposed asialo ACT significantly reduced 3H-thymidine incorporation into human peripheral lymphocytes stimulated by phytohemagglutinin (PHA) though native ACT could not inhibit the mitogen-induced lymphoproliferation and asialo ACT moderately inhibited it. In order to determine the interaction of ACT and its derivatives to lymphocyte membranes, the binding of 125I-labelled ACT and its derivatives to membranes of intact lymphocyte and extracted lymphocyte membranes was examined. The binding of 125I-labelled native ACT and asialo ACT to resting and PHA-stimulated lymphocyte membrane was low. And the binding of 125I-labelled acid-exposed asialo ACT to resting lymphocyte membrane was also low. However, when lymphocytes were stimulated by mitogens the binding of 125I-labelled acid-exposed asialo ACT increased significantly. The binding of 125I-labelled acid-exposed asialo ACT to the membrane extracted from PHA-stimulated lymphocytes was time-dependent and saturation was reached at 120 min at 37 degrees C. One mg of membrane could bind a maximum of approximately 83 pmol of acid-exposed asialo ACT with dissociation constant of 0.73 microM. Other unlabelled serum glycoproteins such alpha-1-antitrypsin, alpha-1-acid glycoprotein, transferrin, and proteinase inhibitors including chymostatin, leupeptin and soy bean trypsin inhibitor did not compete with 125I-labelled acid-exposed asialo ACT for binding sites in simultaneous competition assays.

Comparison of argininosuccinate synthetase from young and old rat livers.

Properties of argininosuccinate synthetases (ASS), including immunological properties and heat stability, from young and old rats were quite similar. However the degradation rates of ASS from young and old rats were found to be different as shown in the experiment using the double labeling technique. Three forms of this enzyme from young and old rat livers were separated by DEAE-Sephadex A-50 column chromatography, and they were called ASS 1, 2 and 3 in order of elution. The degradation rate of ASS 1 from young and old rat livers was very similar, but the rate of degradation of ASS 2 form old rat livers was greater than that from young rat livers.

Incorporation of alpha-1-antichymotrypsin into human stomach adenocarcinoma cell nuclei and inhibition of DNA primase activity.

Incorporation of alpha-1-antichymotrypsin (ACT) into human stomach adenocarcinoma cell nuclei and the effect of ACT on DNA primase from the same carcinoma cells were studied. ACT or [125I]-ACT were observed in carcinoma cell nuclei and high specific radioactivity was detected in washed nuclear fraction when 0.4 mg of ACT or [125I] ACT (8 x 10(7) cpm) was intravenously injected into carcinoma bearing nude mice 2 h before killing. The molecular weight of radioactivity presented in cell nuclei was same as the intact ACT on SDS-polyacrylamide gel electrophoresis. ACT inhibited DNA primase activity and this inhibiting activity was stable than its chymotrypsin inhibiting activity. The results presented here show ACT is incorporated into carcinoma cell nuclei without modification of its molecular weight and may inhibit DNA primase activity.

Inhibition of DNA synthesis by alpha-1-antichymotrypsin.

The effect of alpha-1-antichymotrypsin (ACT), which is known as an efficient serum protease inhibitor and is detected in tumor cell nuclei, on DNA synthesis was studied. ACT inhibited the activity of DNA polymerase alpha purified from human stomach adenocarcinoma. Other human serum proteins including serum albumin, alpha-1-acidglycoprotein, alpha-1-antitrypsin, and immunoglobulin G, as well as other protease inhibitors, such as leupeptin, pepstatin, PMSF and chymostatin, did not affect the activity of DNA polymerase alpha. It was therefore concluded that the inhibitory action of ACT on DNA polymerase alpha was direct phenomenon unrelated to its protease inhibitory activity. Furthermore, the effect of ACT on DNA synthesis was also studied using lysolecithin-permeabilized cultured human stomach carcinoma cells. ACT added in the medium inhibited DNA synthesis and the degree of inhibition depended on incubation time. It was proportional to ACT concentration and the concentration of ACT required for 50% inhibition was 0.8 mg/ml.

Purification, properties and identification of a serum DNA binding protein (64DP) and its microheterogeneity.

A DNA binding protein with a molecular weight of 64,000, designated 64DP, has been purified and characterized. This protein was isolated from adult human pooled serum by DEAE Sephadex column Chromatography, DNA cellulose affinity column chromatography, ammonium sulfate fractionation and Sephadex G-150 gel filtration. The final preparation of 64DP was homogeneous, as judged from polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate and sedimentation experiments. Physicochemical and immunochemical properties of this protein were very similar or identical to those of alpha-1-antichymotrypsin with some differences in electric mobility and th pattern of isoelectric focusing. Furthermore, the general properties of 64DP from various sera were practically similar with the exception that isolectric focusing analysis showed microheterogeneity among 64DP purified from various sera.