hymecromone and Cystic-Fibrosis

In the present study, a structured-based virtual screening (VS) of differently substituted furocoumarins and analogues has been carried out against nuclear factor kappa B (NF-κB), with the objective of selecting molecules able to inhibit the binding of this transcription factor to the DNA. The focus library was developed starting from chemical structures obtained from the literature, as well as retrieving compounds from available commercial databases. A two dimensional substructure searching method based on four different chemical scaffolds was used for this purpose. Among the 10 highest-scored ligands selected from the docking studies, five commercially available molecules were investigated in biological assays. Four furocoumarin derivatives showed IC(50) values in the range of 40-100 μM in inhibiting NF-κB/DNA interactions studied by electrophoretic mobility shift assay (EMSA). Three compounds significantly inhibited NF-κB dependent biological functions (expression of IL-8) in cellular analysis based on Pseudomonas aeruginosa infection of cystic fibrosis IB3-1 cells. These findings validated the virtual screening approach here presented and reinforce the successful results of our previously computational studies aimed at the identification of molecules targeting NF-κB. The discovered novel compounds could be of relevance to identify more potent inhibitors of NF-κB dependent biological functions beneficial to control lung inflammation occurring in patients affected by cystic fibrosis.

Topics: Binding Sites; Cell Line; Computer Simulation; Cystic Fibrosis; Databases, Factual; Electrophoretic Mobility Shift Assay; Furocoumarins; Humans; Hydrogen Bonding; Interleukin-8; Ligands; NF-kappa B; Protein Structure, Tertiary

The ability of cells to decorate glycosaminoglycans (GAGs) with sulphate in highly specific patterns is important to extracellular matrix biogenesis and placing appropriate glycosulphated ligands on the cell surface. We have examined sulphate metabolism in two pancreatic duct epithelial cell lines - PANC-1 and CFPAC-1 (derived from a cystic fibrosis patient) with a view to understanding how pancreatic cells utilise intracellular sulphate. [35S]Sulphate uptake was rapid and reached near steady state levels within 10 min. However, the intracellular specific activity of [35S]sulphate for PANC-1 and CFPAC-1 reached only 35 and 10%, respectively, of the medium specific activity at 10 min. Therefore, sulphate appears to reside within two compartments; a rapidly exchangeable sulphate pool (RESP) and a slowly exchangeable sulphate pool (SESP). Reducing chloride in the medium, increased the specific activity of [35S]sulphate within cells and increased the size of the inorganic sulphate pool, suggesting that the RESP was enlarged. Sulphate pools were not different in size between the two cell lines in physiological NaCl. Increasing the size of the sulphate pool had no effect on [35S]sulphate:[3H]glucosamine ratios incorporated into glycosaminoglycans (GAGs); however, stimulating the synthesis of GAGs with 4-methylumbelliferyl-beta-d-xyloside, stably elevated [35S]:[3H] ratios. This was due to higher [35S]sulphate incorporation. [35S]Cysteine contributed less than 0.1% of the cells' sulphate requirements. We conclude that in the face of elevated demand for sulphate, pancreatic cells appear to channel a greater proportion through the RESP.

Topics: Cell Line; Cystic Fibrosis; Epithelial Cells; Extracellular Matrix; Glycosaminoglycans; Humans; Hymecromone; Pancreas; Sodium Chloride; Sulfates

We evaluated four methods purported to distinguish between individuals homozygous or heterozygous for cystic fibrosis (CF) and normal controls: (1) detection of a protein in the serum by isoelectric focusing at pH 8.5, (2) detection of a lectinlike factor in the serum by hemagglutination, (3) isolation of CF-lectin from the serum by affinity chromatography, and (4) measurement of MUGB-reactive proteases in the plasma. The results were disappointing. The detection of CF protein by isoelectric focusing was unreliable; it could be identified in only 46% of heterozygotes and 66% of homozygotes, with a false positive rate of 17%. Detection of a lectinlike factor by hemagglutination was also found to be unreliable and irreproducible. The lectin isolated by affinity chromatography was not specific for the CF gene. No significant differences were found in the MUGB titers of the three populations tested. However, low titers (MU less than 200 nmol/ml) were found in 33% of homozygotes and heterozygotes and in 17% of normal controls. We conclude that none of these methods is suitable for carrier detection in cystic fibrosis.

Topics: Adult; Blood Proteins; Calgranulin A; Cystic Fibrosis; Heterozygote; Homozygote; Humans; Hymecromone; Lectins; Middle Aged

Amniotic fluids were obtained from 19 mothers who had previously given birth to a child with cystic fibrosis. Measurement of methylumbelliferylguanidinobenzoate (MUGB) reactive proteases suggested that all 19 would have unaffected babies. Amongst the first 10 cases to come to term there were 5 infants with cystic fibrosis. It is concluded that MUGB protease titration is not suitable for the early prenatal diagnosis of cystic fibrosis.

Topics: Amniotic Fluid; Cystic Fibrosis; Female; Gestational Age; Humans; Hymecromone; Peptide Hydrolases; Pregnancy; Prenatal Diagnosis; Umbelliferones

Measurement of 4-methylumbelliferyl p-guanidinobenzoate (MUGB)-hydrolyzing activity in the plasma of normal controls, cystic fibrosis (CF) heterozygotes, and CF homozygotes did not support previously reported (35) differences in MUGB-hydrolyzing activity. We identified human plasma albumin as the major source of MUGB-hydrolyzing activity by comparison of our plasma results to those obtained with physiologic concentrations of commercial albumin samples. Substantiating evidence was obtained from gel filtration experiments and correlation of albumin levels in CF plasma with MUGB titers. We found essentially no proteolytic activity towards dinitrophenylprotamine sulfate associated with commercial albumin samples. It appears that the reaction between human albumin and MUGB represents a weak esterase activity, perhaps involving the acylation of a specific site(s) on the protein. Hypoalbuminemia has been documented (8) in some CF patients. Low albumin concentrations, indicated by MUGB titers less than 190 nmole methylumbelliferone/ml plasma, were found in 42% of CF homozygotes, 6% of heterozygotes, and 4% of controls. Gel filtration studies of a normal amniotic fluid supernatant indicated that albumin was the major MUGB-hydrolyzing substance in this fluid. We conclude that MUGB abnormalities are not associated with the basic gene defect in CF and cannot be used as the basis of a test for intrauterine or heterozygote detection.

Topics: Adolescent; Adult; Amniotic Fluid; Child; Child, Preschool; Chromatography, Gel; Cystic Fibrosis; Genetic Carrier Screening; Humans; Hydrolysis; Hymecromone; Middle Aged; Prenatal Diagnosis; Serum Albumin; Umbelliferones

Topics: Clinical Enzyme Tests; Cystic Fibrosis; Heterozygote; Humans; Hymecromone; Peptide Hydrolases; Serum Albumin; Umbelliferones

4-methylumbelliferylguanidinobenzoate (MUGB) reactivity in plasma from patients with cystic fibrosis and in amniotic fluid from pregnancies leading to children with cystic fibrosis, has been reported to be significantly decreased. We have so far been unable to confirm these findings and have therefore reexamined this reactivity using diisopropylfluorophosphate (DEF), another active site titrant of serine proteases. We have shown that MUGB and DFP are reacting with the same molecules in plasma and amniotic fluid. Using crossed immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis of 3H-DFP labelled plasma and amniotic fluid we have obtained strong evidence that the main contribution of MUGB and DFP reactive protein in plasma and amniotic fluid is identical to serum albumin. The use of MUGB reactivity in amniotic fluid in pregnancies at risk for cystic fibrosis must therefore be reconsidered.

Topics: Amniotic Fluid; Cystic Fibrosis; Endopeptidases; Female; Humans; Hymecromone; Isoflurophate; Pregnancy; Prenatal Diagnosis; Serine Endopeptidases; Serum Albumin; Substrate Specificity

In contrast to the original reports by others, the hydrolysis of 4-methylumbelliferylguanidinobenzoate ((MUGB), and active-site titrant of serine proteases, was found not to be significantly different in plasma samples from patients with cystic fibrosis (CF), obligate heterozygotes, and normal controls. Since the steady-state turnover of substrate is the dominant term of MUGB hydrolysis, the assay in its present form does not meet the requirements of an active-site titration. The reproducibility and reliability of the assay are hampered by (1) high blank values due to non-specific hydrolysis, (2) the absence of a defined endpoint of the incubation, (3) the non-linearity of the assay with respect to plasma concentration, (4) the temperature-dependent evolution of esterase activity during incubation, and (5) most importantly the loss of activity during storage of plasma samples. Cleavage of MUGB catalysed by the imidazole ring of histidine accounts for a significant portion of the activity in plasma.

Topics: Adolescent; Adult; Child; Child, Preschool; Cystic Fibrosis; Drug Stability; Enzyme Activation; Female; Histidine; Humans; Hydrolysis; Hymecromone; Kinetics; Male; Methods; Peptide Hydrolases; Umbelliferones

Topics: Amniotic Fluid; Cystic Fibrosis; False Negative Reactions; Female; Humans; Hymecromone; Isoelectric Focusing; Molecular Weight; Peptide Hydrolases; Pregnancy; Prenatal Diagnosis; Reference Values

Topics: Amniocentesis; Amniotic Fluid; Clinical Enzyme Tests; Cystic Fibrosis; False Negative Reactions; Female; Humans; Hymecromone; Peptide Hydrolases; Pregnancy; Pregnancy Complications; Prenatal Diagnosis; Umbelliferones

Topics: Amniocentesis; Amniotic Fluid; Clinical Enzyme Tests; Cystic Fibrosis; False Positive Reactions; Female; Humans; Hymecromone; Peptide Hydrolases; Pregnancy

Previous studies in our laboratory have suggested that measurement of methylumbelliferylguanidinobenzoate (MUGB) reactive proteases in midtrimester amniotic fluid is potentially of value for the intrauterine detection of cystic fibrosis. Thirty-nine pregnancies of obligate heterozygotes of cystic fibrosis and 12 cases where one parent has a sib with cystic fibrosis have been previously monitored by means of quantitative and qualitative measurements of MUGB in amniotic fluid. In all but one case the diagnosis was accurately ascertained in the midtrimester and confirmed after delivery. The measurement of MUGB reactivity in midtrimester amniotic fluid by three different procedures--quantitative analysis, polyacrylamide isoelectric focusing, and column filtration--appears to provide a practical and reliable approach for the intrauterine detection of cystic fibrosis.

Topics: Amniotic Fluid; Cystic Fibrosis; Female; Heterozygote; Humans; Hymecromone; Pregnancy; Pregnancy Trimester, Second; Prenatal Diagnosis

Topics: Amniotic Fluid; Cystic Fibrosis; Electrophoresis, Polyacrylamide Gel; Female; Humans; Hymecromone; Pregnancy; Pregnancy Trimester, Second; Umbelliferones

An arginine esterase activity similar to that observed in plasma has been demonstrated in second trimester and term human amniotic fluid. Like plasma, the protease(s) hydrolyzed esters of arginine, were reactive towards 4-methylumbelliferylguanidinobenzoate (MUGB), a sensitive active site titrant of trypsin-like enzymes, and had a pI of 5.1--5.4. The pH optimum for proteolytic activity was 8.0. This protease activity was inhibited by soybean trypsin inhibitor (STI), benzamidine and (p-nitrophenyl)-p'-guanidinobenzoate (NPGB), and was insensitive to 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK) and p-hydroxymercuribenzoic acid (HMB). Upon gel filtration, two MUGB-reactive fractions were observed, one with an apparent molecular weight of 200,000 and the other, 100,000. Both fractions had arginine esterase activity and appeared to be sensitive to inhibition by STI and benzamidine. The mean MUGB titre value (nmoles of 4-methylumbelliferone released per ml amniotic fluid) for 300 mid-trimester amniotic fluids was 11.40 +/- 2.40 nmoles MU/ml. The mean specific activity was 2.36 +/- 0.41 nmoles MU/mg protein. Two amniotic fluids from pregnancies which delivered children with cystic fibrosis (CF) were analyzed in blind samples sent from other laboratories. The MU titre values obtained were 4.73 and 4.32 with specific activities of 1.24 and 1.30 respectively. A third was identified in our screening program of amniotic fluids obtained from amniocenteses done for the intrauterine detection of genetic abnormalities. The MU titre value was 5.52 nmoles/ml with a specific activity of 1.34. The specific activities of these fluids when compared to the controls were significantly different (p less than 0.001). The mean titre value for 23 term amniotic fluids samples was 8.14 +/- 1.69 nmoles MU/ml. The mean specific activity was 3.37 +/- 0.76 nmoles MU/mg protein. A term amniotic fluid obtained from a woman who delivered a baby with CF showed a markedly reduced level of MUGB reactivity (3.01 nmole/ml). The specific activity was 1.06 which was significantly different from the control term fluids. The MU titre values and specific activities of amniotic fluids obtained from abnormal pregnancies (such as those with neural tube defects, chromosomal abnormalities and polymorphisms, abortions and stillbirths) and fluids with elevated alphafetoprotein and maternal blood contaminants did not significantly vary from the mean control values (Table 3).

Topics: Amniotic Fluid; Arginine; Chloroform; Cystic Fibrosis; Ellagic Acid; Female; Guanidines; Humans; Hydrolysis; Hymecromone; Peptide Hydrolases; Pregnancy; Pregnancy Trimester, Second; Pregnancy Trimester, Third; Prenatal Diagnosis; Umbelliferones

Over 1,000 specimens of midtrimester human amniotic fluid have been analyzed for 4-methylumbelliferylguanidinobenzoate (MUGB) reactivity. The mean specific activity value (nanomoles of methylumbelliferone (MU) formed per milligram of protein) for the control population (1,037) was 2.37 +/- 0.41. The mean specific activity values from a series of specimens of amniotic fluid derived from abnormal pregnancies (chromosomal and biochemical abnormalities, confirmed neural tube defects, abortions/stillbirths) were found not to vary significantly from value in the control population. The mean specific activity value of blood-tinged samples centrifuged to remove the red blood cell contaminants prior to freezing at -20 degrees C did not vary from the value for the control group, in contrast to the value for samples containing hemolyzed red blood cells (1.76 +/- 0.30). The mean specific activity values for all four groups (control, abnormal, spun and unspun) were found to be significantly different (p less than 0.001) from the mean specific activity value of 1.24 +/- 0.12 for the four cystic fibrosis (CF) amniotic fluids. In addition, upon isoelectric focusing of the control and CF fluids on polyacrylamide gels, pH 5-7, subsequently stained for alpha-N-benzoyl-L-arginine ethyl ester activity, the controls gave a pattern of five activity bands with a pl of 5.1 to 5.4, whereas the CF fluids consistently gave only four. These data suggest the potential value of the MUGB-reactive protease assay for the intrauterine detection of cystic fibrosis.

Topics: Amniocentesis; Amniotic Fluid; Cystic Fibrosis; Female; Humans; Hymecromone; Peptide Hydrolases; Pregnancy; Umbelliferones

Thirteen pregnancies of obligate heterozygotes of cystic fibrosis have been prospectively monitored utilizing quantitative and qualitative measurements of methylumbelliferylguanidinobenzoate (MUGB) reactive proteases in amniotic fluid. In each case the diagnosis was accurately ascertained in midtrimester and confirmed after delivery. The measurement of MUGB reactivity in midtrimester amniotic fluid by three different procedures, quantitative analysis, polyacrylamide isoelectric focusing, and column filtration appears to provide a practical and reliable approach for the intrauterine detection of cystic fibrosis.

Topics: Amniotic Fluid; Chromatography, Gel; Cystic Fibrosis; Female; Humans; Hymecromone; Isoelectric Focusing; Peptide Hydrolases; Pregnancy; Prenatal Diagnosis; Umbelliferones

Topics: Amniocentesis; Amniotic Fluid; Cystic Fibrosis; Female; Humans; Hymecromone; Peptide Hydrolases; Pregnancy; Pregnancy Trimester, Second; Umbelliferones

Topics: Amniotic Fluid; Cystic Fibrosis; Female; Humans; Hymecromone; Peptide Hydrolases; Pregnancy

Protease activity in plasma is assayed using 4-methylumbelliferylguanidinobenzoate. The assay is modified by carrying out the reaction in the presence and absence of benzamidine, a competitive inhibitor of trypsin-like proteases. The parameters of the assay are described in detail. Using this assay, our earlier demonstration of a deficiency of protease activity in plasma of patients with cystic fibrosis is confirmed. The activity, corrected for the nonspecific hydrolysis of 4-methylumbelliferylguanidinobenzoate by benzamidine, is expressed as nanomoles of 4-methylumbelliferone released per milliliter plasma. Under standard conditions, the activity in plasma activated with chloroform-ellagic acid was 127.2 +/- 23.1 in 7 controls, 70.4 +/- 11.7 in 11 obligate heterozygotes, and 48.7 +/- 16.6 in 12 patients with cystic fibrosis. Identical results were obtained when unactivated plasma was used. These data demonstrate that the judicious use of specific inhibitors such as benzamidine might be useful in assaying low levels of protease activity in crude systems.

Topics: Benzamidines; Child; Cystic Fibrosis; Guanidines; Humans; Hymecromone; Peptide Hydrolases; Trypsin Inhibitors; Umbelliferones

Protease activity in cultivated human skin fibroblasts has been quantitated using 4-methylumbelliferylguanidinobenzoate (MUGB), an active site titrant of trypsin-like proteases (7). The reaction of the proteases with MUGB was complete in 1 hr, inhibited both by benzamidine and (p-nitrophenyl)-p'-guanidinobenzoate, but not by p-hydroxymercuribenzoate. The extent of reaction was proportional to protein concentration and independent of MUGB concentration. This activity was present in the particulate fraction of the cell. The mean "titre" values (nanomoles of 4-methylumbelliferone released per mg protein) of the proteases in fibroblasts from eight controls (N), 8 obligate heterozygotes (H), and 14 patients with cystic fibrosis (CF) were: N, 1.27 +/- 0.11; H, 0.82 +/- 0.12; CF, 0.66 +/- 0.10. The differences in the "titre" values for N:CF and N:H were significant (p less than 0.001) as were those for H:CF (p less than 0.01). The mean "titre" value obtained for cultivated control amniotic fluid cells was 1.29 +/- 0.17. These data indicate a reduction in the MUGB-reactive proteases in skin fibroblasts derived from patients with CF when compared either to control or to obligate heterozygotes. These data are consistent with our earlier suggestion (11, 15) that decreased proteolytic levels in the tissues and fluids of patients with CF may be a generalized phenomenon.

Topics: Biological Assay; Cells, Cultured; Cystic Fibrosis; Fibroblasts; Guanidines; Humans; Hymecromone; Peptide Hydrolases; Skin; Umbelliferones

Protease activity, assayed using 4-methylumbelliferylguanidinobenzoate, an active site titrant of certain proteases, is significantly deficient in plasma of patients with cystic fibrosis. The deficiency can be demonstrated with both chloroform-ellagic acid activated plasma in which the proteases can hydrolyze esters of arginine and unactivated plasma in which the proteases have negligible activity towards these esters. The deficiency can also be demonstrated by separation of the proteases by isoelectric focusing on polyacrylamide gels or by chromatography on agarose columsn. Since protease deficiency can be demonstrated with unactivated plasma, the deficiency in cystic fibrosis is probably due to a reduced number of protease molecules rather than their decreased catalytic efficiency.

Topics: Adolescent; Arginine; Binding Sites; Child; Child, Preschool; Cystic Fibrosis; Guanidines; Humans; Hymecromone; In Vitro Techniques; Isoelectric Focusing; Molecular Weight; Peptide Hydrolases; Umbelliferones