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J Wolff

Publications and source records attributed to J Wolff.

At least 145 records · Page 8Linked to original sources
PubMed1988

Bordetella pertussis adenylate cyclase. Penetration into host cells.

Exposure of Chinese hamster ovary, mouse adrenal cortex tumor (Y-1), THP-1 and U-937 cells and human erythrocytes to adenylate-cyclase-containing urea extracts of Bordetella pertussis (strain 114) organisms promotes the formation of large concentrations of intracellular cAMP. Accumulation is dependent on dose and temperature, with significant accumulation occurring at 4 degrees C, and is virtually instantaneous, with a doubling at 1 min. There is an absolute Ca2+ requirement but external calmodulin (the activator of cyclase activity) has no effect except in erythrocytes and U-937 cells, where it reduces cAMP accumulation. However, calmodulin antagonists inhibit cAMP accumulation. In Y-1 adrenal cells the urea-extract adenylate cyclase stimulates steroidogenesis. Anti-(B. pertussis) antibodies inhibit cyclase activity and prevent further cAMP accumulation after 10 min in cells previously exposed to urea extract. The same effect is obtained by washing. This suggests that a portion of the cyclase is associated with cells in a form not accessible to antibody or washing but accessible to substrate, which we interpret as internalized enzyme with a short lifetime. Continuing cAMP accumulation thus appears to need a continuing source of external cyclase. Inhibitors of the effect of diphtheria toxin, such as NH4Cl, methylamine, chloroquine or monensin, have no inhibitory effect on the accumulation of intracellular cAMP promoted by the internalized adenylate cyclase of urea extracts of B. pertussis organisms. We conclude that entry of the cyclase into cells is not by receptor-mediated endocytosis.

Adenylate Cyclase Toxin↗
PubMed1988

Structure and energetics of ligand binding to proteins: Escherichia coli dihydrofolate reductase-trimethoprim, a drug-receptor system.

A study of the binding of the antibacterial agent trimethoprim to Escherichia coli dihydrofolate reductase was carried out using energy minimization techniques with both a full, all-atom valence force field and a united atom force field. Convergence criteria ensured that no significant structural or energetic changes would occur with further minimization. Root-mean-square (RMS) deviations of both minimized structures with the experimental structure were calculated for selected regions of the protein. In the active site, the all-atom minimized structure fit the experimental structure much better than did the united atom structure. To ascertain what constitutes a good fit, the RMS deviations between crystal structures of the same enzyme either from different species or in different crystal environments were compared. The differences between the active site of the all-atom minimized structure and the experimental structure are similar to differences observed between crystal structures of the same protein. Finally, the energetics of ligand binding were analyzed for the all-atom minimized coordinates. Strain energy induced in the ligand, the corresponding entropy loss due to shifts in harmonic frequencies, and the role of specific residues in ligand binding were examined. Water molecules, even those not in direct contact with the ligand, were found to have significant interaction energies with the ligand. Thus, the inclusion of at least one shell of waters may be vital for accurate simulations of enzyme complexes.

Computer Simulation↗
PubMed1988

Influence of renal function on the elimination of morphine and morphine glucuronides.

The influence of renal function, measured by 51Cr-EDTA clearance, on morphine and morphine glucuronide kinetics has been studied in 13 patients after a single i.v. injection of morphine. Unconjugated morphine and morphine glucuronides were measured by a sensitive, specific RIA after extraction from plasma. No significant correlation was found between total body clearance of unconjugated morphine and 51Cr-EDTA clearance. However, patients with renal insufficiency had impaired elimination of morphine glucuronides, and the apparent clearance was significantly correlated with the 51Cr-EDTA clearance (r = 0.94, p less than 0.001). A relatively long terminal elimination of half-life of morphine was found in all patients (mean +/- SD: 9.2 +/- 2.5 h), irrespective of glomerular function.

Aged↗
PubMed1988

[Chemical and toxicologic studies of native corn in flour and bread].

During the bread-baking process, total alkaloid content is reduced by about 50%, the pharmacologically active alkaloids (the '-in' types) being reduced to a much greater extent than the less active type ('-inin' type). Using ergotamin and ergokryptin as a basis, total alkaloid levels were calculated from the HPLC data; these results were in a good agreement with the total alkaloid content calculated from 13 individual ergot alkaloids. The changes in the alkaloid spectrum caused by pelletin are shown. In feeding experiments with growing, pregnant, and lactating mice, diets with 0%, 1% and 2% unbaked (MM) or baked (MB) rye ergot were compared for possible toxic effects on reproduction, using animals fed ad libitum or pair-fed controls. A mean ingestion of 3.6 mg of alkaloids/kg body weight per day (MM 2%) was maternally toxic and caused inhibition of conception and implantation. Feeding with 1.3 or 1.6 mg of alkaloids/kg body weight per day (MM 1% and MB 2%) during pregnancy had no significant toxic effect on the embryo or fetus, but resulted in gestation time being prolonged to about 10 h. During lactation, an intake of 2.9 and 3.0 mg of alkaloids/kg body weight per day (MM 1% and MB 2%) led to a considerable reduction in growth (up to 50%) in the young mice compared to the control animals fed ad libitum. Both the determination of total ergot alkaloid content and the feeding experiment indicate that the bread-baking process causes a reduction and consequently a detoxification of ergot alkaloids of about 50%.

Abnormalities, Drug-Induced↗
PubMed1988

Pharmacokinetics of midazolam and alpha-hydroxy-midazolam following rectal and intravenous administration.

1. In an open cross-over trial, plasma concentrations of midazolam were measured in eight healthy male volunteers following administration of 0.3 mg kg-1 body weight given by the rectal and intravenous routes. 2. Maximum plasma concentrations of 92-156 ng ml-1 (mean 118 ng ml-1) were recorded from 20 to 50 min (mean 31 min) after rectal application. The rectal bioavailability was 40-65% (mean 52%) and the terminal half-life was 114-305 min (mean 161 min). 3. A substantial first-pass hepatic effect was observed following rectal administration. 4. No systemic or local intolerance was noted. 5. In conclusion, the rectal route of administration provides a rapid and reliable absorption of midazolam.

Administration, Rectal↗
PubMed1987

Nile red as a polarity-sensitive fluorescent probe of hydrophobic protein surfaces.

Nile red is an uncharged hydrophobic molecule whose fluorescence is strongly influenced by the polarity of its environment. It interacts with many, but not all, native proteins, including beta-lactoglobulin, kappa-casein, and albumin, with a wide range of spectral changes for different proteins. It detects the exposure or formation of new hydrophobic surfaces induced by ligand binding to calmodulin, oligomerization of melittin, or unfolding of ovalbumin during early thermal denaturation. The dye is photostable, the working wavelength range is broad and removed from those at which many biomolecules absorb, the fluorescence is unaffected by pH between 4.5 and 8.5, the quantum yield is high, and hydrophobic sites on proteins may be investigated in dilute solutions.

Binding Sites↗
PubMed1987

Lipoprotein patterns in nondiabetic, borderline diabetic, and diabetic Macaca nigra.

Lipoproteins were isolated by sequential ultracentrifugation, and the concentrations and compositions were determined in nondiabetic (ND), borderline diabetic (BD), and diabetic (D) Macaca nigra males consuming a chow ration. The total concentrations and components of the VLDL and IDL increased significantly with metabolic deterioration (P less than 0.01). Concentrations and components of LDL increased in the BD and D monkeys, but changes were not statistically significant. The HDL2 and HDL3 particles were virtually unchanged among the three different metabolic groups. The VLDL was the major carrier of the triglycerides, especially in D monkeys. Cholesterol was present predominantly in the LDL. The LDL-cholesterol to HDL-cholesterol ratio increased in the BD and D monkeys, owing mainly to increases in the LDL-cholesterol content. Apoprotein antisera showed apoprotein B in the VLDL, IDL, and LDL, apoprotein E in the VLDL and IDL, and apoprotein A-I in the HDL2 and HDL3 fractions. Because Macaca nigra consume a nonatherogenic, low-cholesterol, low-fat ration, the changes in lipoproteins, particularly in VLDL and IDL, are attributable to metabolic alterations associated with diabetes.

Animals↗
PubMed1987

Epitope insertion into the human hypoxanthine phosphoribosyltransferase protein and detection of the mutant protein by an anti-peptide antibody.

The translational stop codon TAA of the human hypoxanthine phosphoribosyltransferase (HPRT) cDNA has been changed to GAA by site-specific mutagenesis. This modification extends the open reading frame to a downstream stop codon and results in the addition of a unique negatively charged hexapeptide to the C terminus of human HPRT protein. The mutated cDNA was transferred into HPRT-deficient rodent cells by retroviral vector infection, and the expressed enzyme was found to be fully active. An antibody against a synthetic octapeptide corresponding to the mutated HPRT C terminus precipitated the HPRT protein specifically from cells infected with the mutant virus and not infected with the wild-type HPRT virus. The technique of inserting a novel epitope into a protein by site-directed mutagenesis should be generally applicable in studies of the regulation of gene expression in vitro and in vivo.

Epitopes↗
PubMed1987

Pharmacologic amino acid acylation in the acute hyperammonemia of propionic acidemia.

An infant with propionic acidemia presented at one month of age with hyperammonemic coma. Treatment by two double-volume exchange transfusions did not have an appreciable effect, but hemodialysis led to a substantial reduction of the serum concentration of ammonia on two occasions. Nevertheless, continued therapy with sodium benzoate, arginine-HCl, carnitine and lactulose did not have any observable effect on the blood concentration of ammonia. Treatment with sodium phenylacetate was followed by a reduction in serum concentrations of ammonia to normal levels which were maintained. These observations demonstrate the dramatic reduction in serum concentrations of ammonia that may be obtained in patients with organic acidemia by hemodialysis. They suggest that pharmacologic acylation therapy with phenylacetate may be of lasting benefit in the management of this complication.

Acylation↗
PubMed1986

Activation of Bordetella pertussis adenylate cyclase by the carboxyl-terminal tryptic fragment of calmodulin.

Highly purified tryptic fragments of calmodulin were tested for their ability to stimulate adenylate cyclase activity of Bordetella pertussis spheroplast membranes and were compared to their activities on brain Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase. The C-terminal fragment, consisting of residues 78-148, was a full agonist for the cyclase with 0.1-0.15 the potency of calmodulin but did not stimulate phosphodiesterase. Fragments 1-77, 1-90, and 107-148 stimulated adenylate cyclase (and not phosphodiesterase) at low potency; this was not due to calmodulin contamination, but contamination by fragment 78-148 could not be excluded with certainty. An adduct of norchlorpromazine isothiocyanate and calmodulin showed full agonist activity for adenylate cyclase at 0.01-0.02 the potency of calmodulin. Stimulation of adenylate cyclase by a number of the fragments occurred in the absence of Ca2+, but stimulator potency was enhanced 20-60-fold in its presence. The similarity of Ca2+ requirements of fragment 78-148 and calmodulin suggests that occupancy of the two C-terminal Ca2+ binding sites of calmodulin accounts for most of the Ca2+ enhancement of calmodulin stimulation of adenylate cyclase.

Adenylyl Cyclases↗
PubMed1986

Cyclic AMP-independent stimulation of steroidogenesis in Y-1 adrenal tumor cells by antimitotic agents.

The stimulation of steroidogenesis by antimitotic drugs has been studied in wild-type (Y-1) and cAMP-dependent protein kinase-deficient (kin-8) mouse adrenal tumor cell lines. Unlike some other cells, Y-1 cells do not increase their cAMP output upon exposure to antimitotic drugs such as colchicine, vinblastine or podophyllotoxin, which readily increase steroidogenesis. Moreover, no increase in cAMP can be detected over an extended time span. Stabilization of tubulin polymers by taxol or high concentrations of vinblastine blocks ACTH-, cholera toxin- or colchicine-stimulated steroidogenesis without major effects on cAMP levels. Colchicine and podophyllotoxin stimulate steroidogenesis in the cAMP-dependent protein kinase-deficient mutant to the same degree as in the wild-type Y-1 cells, although absolute steroid yields are lower in the mutant cells. We suggest that the antimitotic agents stimulate adrenal steroidogenesis by a cAMP-independent pathway that may involve facilitation of cholesterol access to the mitochondrion.

Adrenal Gland Neoplasms↗