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Biomedical subjects

J Hepp

Publications and source records attributed to J Hepp.

At least 37 records · Page 2Linked to original sources
PubMed1987

Covalent labeling of opioid receptors with 3H-D-Ala2-Leu5-enkephalin chloromethyl ketone. I. Binding characteristics in rat brain membranes.

The chloromethyl ketone derivative of D-Ala2-Leu5-enkephalin was synthesized in a radioactive form, and the resulting compound (3H-DALECK) was used to label opioid receptors. 3H-DALECK binds with high affinity, specificity and saturability to rat brain membranes. The number of sites labeled is 130 fmoles/mg protein. Unlabeled opioids inhibited the binding of 3H-DALECK; etorphine and DAGO being most potent. A 10-fold preference for mu sites over delta was seen in site-specific competition experiments; while DALECK displayed low affinity for kappa sites of rat brain. DALECK irreversibly blocked a certain population of sites. Approximately 40% of 3H-DALECK binding at 15 min, and 60% at 60 min association time did not dissociate in the presence of a large excess of unlabeled DALECK and was resistant to washing. Autoradiography performed after SDS-PAGE revealed specific alkylation of proteins with molecular weight of 74, 65, 56, 43 and 34 kD. These results demonstrate the applicability of using 3H-DALECK to covalently label opioid receptors.

Amino Acid Chloromethyl Ketones↗
PubMed1987

Covalent labeling of opioid receptors with 3H-D-Ala2-Leu5-enkephalin chloromethyl ketone. II. Binding characteristics in frog brain membranes.

3H-D-Ala2-Leu5-enkephalin chloromethyl ketone (3H-DALECK) was used to label opioid receptors of frog brain membranes. We have previously shown (15) that 70% of the opioid receptors are of kappa type in this preparation. The binding of 3H-DALECK was of high affinity, half maximal binding being achieved by 0.9 nM of the radioligand. The number of sites labeled was calculated to be 108 fmol/mg protein. Opioid ligands, incubated with the membranes prior to the label, inhibited 3H-DALECK binding with the following rank order:etorphine greater than EKC greater than DAGO greater than DALECK greater than DADLE. Dissociation experiments showed that 70% of the binding is irreversible. Fluorography performed after SDS-PAGE revealed specific covalent labeling of protein subunits of 90, 58 and 20 kD molecular weights. Results will be compared to those obtained in rat brain (13). Our two studies demonstrate that 3H-DALECK is a useful probe for investigation the subunit structure of opioid receptors.

Amino Acid Chloromethyl Ketones↗
PubMed1987

Tyr-D-Ala-Gly-(Me)Phe-chloromethyl ketone: a mu specific affinity label for the opioid receptor.

An alkylating tetrapeptide enkephalin derivative, Tyr-D-Ala-Gly-(Me)Phe-chloromethyl ketone (DAMK) was synthesized, and its binding characteristics on rat brain membranes were evaluated. In competition experiments, the product shows high affinity for the mu opioid binding site of the rat brain membranes, whereas its binding to the delta and kappa subtypes is weak. Micromolar concentrations of this ligand produce a dose-dependent, apparently irreversible inhibition of /3H/-naloxone binding, with apparent IC50 value of 1-5 uM. Neither reversibly binding opioids nor tosyl-amino acid chloromethyl ketones show these effects. Saturation binding analysis with /3H/-naloxone of membranes preincubated with Tyr-D-Ala-Gly-(Me)Phe-CH2Cl reveal a selective and irreversible inhibition of the high affinity /3H/-naloxone binding site. Irreversible blockade of mu-selective /3H/-ligand binding by Tyr-D-Ala-Gly-(Me)Phe-CH2Cl is much more effective than that of the binding of /3H/-enkephalin or /3H/-ethylketocyclazocine. The mu-selective binding properties of this new irreversible enkephalin analogue suggest that it could serve as an affinity label for the mu opioid receptor subtype.

Affinity Labels↗
PubMed1987

Purification of a kappa-opioid receptor subtype from frog brain.

A kappa-opioid receptor subtype was purified from a digitonin solubilized preparation of frog brain membranes using affinity chromatography. The affinity resin was prepared by coupling D-Ala2-Leu5-enkephalin to Sepharose-6B matrix. After elution of the receptor by 50 mumol naloxone, the kappa-subtype was separated from the mu- and delta-subtypes by gel permeation chromatography on Sepharose-6B. The purified receptor binds 3,900 pmol [3H]-ethylketocyclazocine per mg protein (a 4,300-fold purification over the membrane-bound receptor) with a KD of 8.3 nM. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows two bands (Mr 65,000 and 58,000) in sodium dodecyl sulfate gel electrophoresis.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1978

Long term results of Roux-en-Y hepaticojejunostomy.

One hundred and twenty-three patients with benign diseases involving the bile ducts, curable by biliary bypass, underwent a Roux-en-Y hepaticojejunostomy, and the long term results of this operation were evaluated to ascertain if hepaticojejunostomy is a safe and durable procedure. There were no operative deaths. A peptic ulcer developed postoperatively in two patients. Only one instance of anastomotic stenosis was observed at follow-up study, averaging 5.5 years. Six of the 11 patients with postoperative biliary symptoms had intrahepatic lithiasis, a possible indication that the symptoms were due to temporary obstruction of the anastomosis by residual stones. A refined operative technique has improved the results which over-all are highly satisfactory.

Adult↗
PubMed1977

Improving cholecystectomy.

Bacteriologic study of bile in 100 patients undergoing cholecystectomy for various manifestations of choletithiasis yielded 36 per cent positive cultures, with greater frequency in older individuals and those with acute cholecystitis and common duct stone; these results are comparable to those in previous studies and reaffirm the septicity of the bile. Incidence of wound infection, averanging 10 per cent in published series of cholecystectomies, was 0.5 per cent in 200 patients in whom a water-impermeable wound drape was sewn to the peritoneum to prevent contamination by potentially infected bile. This result, in patients with an infectious risk comparable to that in other series, establishes the value of meticulous wound isolation in preventing wound infection.

Adult↗