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Biomedical subjects

J Hepp

Publications and source records attributed to J Hepp.

At least 19 recordsLinked to original sources
PubMed1997

Affinity labelling of frog brain opioid receptors by dynorphin(1-10) chloromethyl ketone.

It has been previously found that chloromethyl ketone derivatives of enkephalins bind irreversibly to the opioid receptors in vitro. Recently a novel affinity reagent, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Gly chloromethyl ketone (Dynorphin(1-10)-Gly11 chloromethyl ketone, DynCMK) was synthesized, and its binding characteristics to frog (Rana esculenta) brain membranes were evaluated. In competition experiments, the product shows a relatively high affinity for the kappa-opioid binding sites labelled by [3H]ethylketocyclazocine (Ki is approximately equal to 200 nM), whereas its binding to the 1 ([3H]dihydromorphine) and to the delta sites ([3H]D-Ala2-Leu5]enkephalin) is weaker. Preincubation of the frog brain membranes with DynCMK at micromolar concentrations results in a washing-resistant and dose-dependent inhibition of the [3H]ethylketocyclazocine binding sites. Saturation binding analysis of the membranes preincubated with 50 microM DynCMK reveals a significant decrease in the number of specific binding sites for [3H]ethylketocyclazocine compared to the control values. The kappa-preferring binding properties of the compound suggest that it could serve as an affinity label for the kappa-type of opioid receptors.

Affinity Labels↗
PubMed1997

[Chronic pancreatitis. A recent national experience with its surgical management].

BACKGROUND: The prevalence of chronic pancreatitis in Chile is not known and there is no local information about the surgical treatment of this disease. AIM: To review retrospectively the results of surgical treatment of chronic pancreatitis. MATERIAL AND METHODS: The charts of 17 patients (12 male), aged 7 to 65 years old, with chronic pancreatitis that were operated in three different Chilean regions, were reviewed. RESULTS: Seven patients had previous endoscopic therapeutic procedures (papillotomy in 4 and stent placing in 3). Seven patients had been subjected to previous biliary surgical procedures. Indications for surgery were severe pain in 14 patients, the suspicion of a pancreatic carcinoma in 4 patients, an infected pseudocyst in one and massive bleeding of multiple pseudo-aneurysms in a pseudocyst in one patient. Twelve patients were subjected to decompressions and 5 to pancreatic resections. There was no operative mortality and one transient pancreatic fistula. After an average follow up of 22 months, pain improved in 94% of cases, pancreatic cancer was diagnosed in one patient and 79% of subjects gained weight. One patient became insulin dependent, one increased his insulin requirements and one had transient steatorrhea, since she could not afford pancreatic enzyme replacement therapy. CONCLUSIONS: The multidisciplinary approach of patients with chronic pancreatitis, with selective use of surgery, may greatly improve their quality of life.

Adolescent↗
PubMed1995

Substrate- and inhibitor-specificity of a non-endothelial enzyme which forms [Met5]-enkephalin from [Met5]-enkephalin-Arg6,Phe7 in isolated rabbit ear artery: pharmacological characterization.

The captopril-inhibited enzyme which forms [Met5]-enkephalin from [Met5]-enkephalin-Arg6,Phe7 in isolated rabbit ear artery was characterized further by using various natural substrate candidates/analogues ([Met5]-enkephalin-Arg6,Phe7 and its amide, [Met5]-enkephalin, angiotensin I and bradykinin), peptidase inhibitors such as captopril, enalaprilate and thiorphan and by endothelial removal. 10(-5) and 10(-4) M but not 10(-6) M captopril reduced the effectiveness of [Met5]-enkephalin-Arg6,Phe7 and potentiated the effect of bradykinin but did not affect markedly the action of the other peptides. Of the inhibitors, enalaprilate was less effective than captopril, and thiorphan had no effect. The [Met5]-enkephalin-Arg6,Phe7-->[Met5]-enkephalin conversion was not affected by endothelial removal. The substrate and inhibitor spectrum of this non-endothelial enzyme activity bears no relationship in other, hitherto characterized dipeptidylcarboxypeptidases/endopeptidases known to be involved in the metabolism of the tested peptides.

Amino Acid Sequence↗
PubMed1994

Design of opioid peptides for a potential delta-receptor affinity label function: comparison with the mu-specific Tyr-D-Ala-Gly-(Me)Phe-chloromethyl ketone.

To find a delta-opioid receptor preferring peptide structure containing an Asp residue in a potentially interacting position, Tyr-Pro-Phe-Asp, Tyr-D-Ala-Phe-Asp, Tyr-D-Ala-Gly-Phe-Asp, Tyr-D-Ala-Gly-Phe-Asp alpha- and beta-methyl ester and Tyr-Gly-Gly-Phe-Asp peptides were synthesized and their biological activities were analyzed in vitro in mouse vas deferens and longitudinal muscle strip of guinea pig ileum. Changing the beta-methyl ester for an alkylating chloromethyl ketone moiety in the delta-receptor-selective agonist Tyr-D-Ala-Gly-Phe-Asp-beta-methyl ester enhanced further the delta-receptor preference. The delta-receptor selective chloromethyl ketone but not the beta-methyl ester gave a very slow washout after prolonged incubation in the mouse vas deferens bioassay; however, it was still readily displaceable by naloxone. The washout pattern of mu-specific Tyr-D-Ala-Gly-(Me)Phe chloromethyl ketone did not differ in the bioassays from that of the corresponding Gly5-ol derivative. Both chloromethyl ketones gave irreversible characteristics in the receptor binding assay.

Affinity Labels↗
PubMed1992

A novel opioid structure which accepts protonated as well as non-protonated nitrogen: a family of pure, delta receptor selective antagonists.

Conventional opioids including opioid peptides require an "opioid" nitrogen which exists in protonated state while interacting with the receptor. In the present paper we demonstrate that the Tyr-Pro-Gly-Phe-Leu-Thr hexapeptide sequence accepts N-terminal substituents such as N-t-Boc, N-phenylacetyl and N-diphenylacetyl where the N cannot become protonated, as well as "traditional" substitutions such as N,N-diallyl, where protonation is likely under physiological conditions. The opioid peptides bearing these substituents are pure antagonists of medium affinity (Ke values in the mouse vas deferens bioassay against [Met5]-enkephalin are in the 3 x 10(-7)-4 x 10(-6) M range) with a high delta receptor preference (50-350-fold delta over mu selectivity ratios).

Amino Acid Sequence↗
PubMed1992

[A giant hepatic hemangioma and pregnancy].

We present a clinical case of a pregnancy with giant hepatic hemangioma. At term it presented like a consumption coagulopathy with thrombocytopenia. Maternal and fetal color Doppler was normal. The perinatal outcome was successful.

Adult↗
PubMed1991

Opioid receptor labeling with the chloromethyl ketone derivative of (3)H-Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAMGO) I: Binding properties of 3H-Tyr-D-Ala-Gly-(Me)Phe chloromethyl.

We prepared a tritiated chloromethyl ketone derivative of Tyr-D-Ala-Gly(Me)Phe-Gly-ol 3H-D-Ala-Gly-(Me)Phe-chloromethyl ketone, and studied its binding characteristics in rat brain membranes. A significant portion (about 70%) of the binding becomes wash-resistant after 60 min of incubation. The binding of the ligand is highly stereospecific and mu-opioid receptor selective. These characteristics of the ligand, together with its high specific radioactivity (57 Ci/mmol) makes it a good candidate for biochemical characterization and covalent labeling of mu opioid receptors.

Amino Acid Chloromethyl Ketones↗
PubMed1990

Method for isolation of kappa-opioid binding sites by dynorphin affinity chromatography.

A kappa-opioid receptor subtype was purified from a digitonin extract of frog brain membranes, using affinity chromatography. The affinity resin was prepared by coupling dynorphin (1-10) to AH Sepharose 4B. The purified receptor binds 4,750 pmol [3H]ethylketocyclazocine (EKC) per mg protein (5,600-fold purification over the membrane-bound receptor) with a Kd of 9.1 nM. The addition of cholesterol-phosphatidylethanolamine (2:1) enhanced 3.6-fold the binding activity of the purified material, which gives a purification very close to the theoretical. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows one major band (65,000 Mr) in sodium dodecyl sulfate (SDS) gel electrophoresis.

Animals↗
PubMed1990

Characterization of kappa 1 and kappa 2 opioid binding sites in frog (Rana esculenta) brain membrane preparation.

The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappa 1 is the dominant receptor subtype, frog brain contains mainly the kappa 2 subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition experiments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazidomorphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa 2 ligands (Met5)enkephalin-Arg6-Phe7 and etorphine also show high affinities to the frog brain. Kappa 1 binding sites measured in the presence of 5 microM/D-Ala2-Leu5/enkephalin represent 25-30% of [3H]ethylketocyclazocine binding in frog brain membranes. The kappa 2 subtype in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and /D-Ala2-(Me)Phe4-Gly5-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain.

Amino Acid Sequence↗
PubMed1990

Dipeptidyl peptidase IV (DP IV) and superoxide dismutase activity in thymus-derived lymphocytes: effects of inhibitory peptides and Zn2+ in vitro.

DP IV and superoxide dismutase (SOD) activity in thymus-derived lymphocytes of rats was assayed in vitro. The DP IV activity was measured fluorimetrically by hydrolysis of Leu-Pro-AMC, and the SOD activity by the inhibition of autooxidation of L-adrenaline. In order of their competitive inhibitory potency the following peptides were tested against DP IV and SOD activity: Ile-Pro-Ile (diprotin A), Val-Pro-Leu (diprotin B), Ile-Pro, Leu-Pro, Val-Pro, Tyr-D--Ala-Ala-Pro, Phe-Pro, Tyr-Pro, Ala-Ala-Pro and Gly-Pro. The peptides, in the order of their potency against DP IV, were effective to inhibit the SOD activity in T lymphocytes. Zn2+ ions exerted an inhibition on both DP IV and SOD activity in a near equimolar concentration. The involvement of Zn2+ as well as the peptides liberated by hydrolysis of polypeptides in regulation of cell-mediated immune responses has been discussed.

Amino Acid Sequence↗
PubMed1990

[Perspectives of liver transplantation in Chile].

After an initial experience with 4 patients who received orthotopic liver transplant (OLT) we analyze the perspectives of establishing liver transplantation programs in Chile. An estimated 100 adult and pediatric patients require OLT a year and no more than 3 centers are required to deal with this and acquire experience with the procedure. A national organization for organ procurement is essential, given the marked difficulties in obtaining cadaver organs at present. Each procedure has an estimated cost of $5 million with 2 more millions in the first year. Thus a program for 15 patients a year may cost $ 100 million, personnel expenses not included.

Adult↗
PubMed1989

Changes of dipeptidyl peptidase (DP IV) activity in the T lymphocytes of rats following administration of ACTH, dexamethasone and opiates.

The aim of the work was to study the effect of glucocorticoids, opiates and stressful stimuli on dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) activity of T lymphocytes prepared from the thymus of intact and adrenalectomized rats. Four week old male rats of Wistar strain were used. The in vivo administration of ACTH, dexamethasone and morphine treatment resulted in an increase of DP IV activity in the cell suspension. In adrenalectomized rats ACTH treatment failed to modify the enzyme activity, however, pain or emotional stress resulted in an elevated DP IV activity. Morphine and D-Met2-Pro5-enkephalinamide resulted in a dose dependent activation of DP IV in T cells, an effect which could be modified by naloxone pretreatment. Our findings show that DP IV mechanisms in T cells are highly sensitive to exogenous and endogenous steroids, opiates and biologically active substances released in response to stress in rats.

Adrenocorticotropic Hormone↗
PubMed1988

Synthesis of 3H-Tyr-D-Ala-Gly-N(Me)Phe chloromethyl ketone--an opioid affinity label.

A radioactive enkephalin affinity reagent, selective for the mu opioid receptor subtype, was synthesized by a fragment condensation method. 3H-BOC-Tyr-D-Ala-Gly-OH was prepared by catalytic tritiation of the protected iodinated tripeptide. The protected tritiated tripeptide and N(Me)Phe-CH2Cl were condensed by the mixed anhydride method. The protecting group was removed by HCl/acetic acid. The tritiated tetrapeptide has a specific radioactivity of 56.8 Ci/mmole (2.1 TBq/mmole).

Affinity Labels↗
PubMed1988

Electrostatic complementarity within the substrate-binding pocket of trypsin.

The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replaced by serine (present in a similar position in chymotrypsin) through site-directed mutagenesis. The wild-type (with Asp-189 in the mature trypsin sequence) and mutant (Ser-189) trypsinogens were expressed in Escherichia coli, purified to homogeneity, activated by enterokinase, and tested with a series of fluorogenic tetrapeptide substrates with the general formula succinyl-Ala-Ala-Pro-Xaa-AMC, where AMC is 7-amino-4-methyl-coumarin and Xaa is Lys, Arg, Tyr, Phe, Leu, or Trp. As compared to [Asp189]trypsin, the activity of [Ser189]trypsin on lysyl and arginyl substrates decreased by about 5 orders of magnitude while its Km values increased only 2- to 6-fold. In contrast, [Ser189]trypsin was 10-50 times more active on the less preferred, chymotrypsin-type substrates (tyrosyl, phenylalanyl, leucyl, and tryptophanyl). The activity of [Ser189]trypsin on lysyl substrate was about 100-fold greater at pH 10.5 than at pH 7.0, indicating that the unprotonated lysine is preferred. Assuming the reaction mechanisms of the wild-type and mutant enzymes to be the same, we calculated the changes in the transition-state energies for various enzyme-substrate pairs to reflect electrostatic and hydrogen-bond interactions. The relative binding energies (E) in the transition state are as follows: EII greater than EPP greater than EPA greater than EIP approximately equal to EIA, where I = ionic, P = nonionic but polar, and A = apolar residues in the binding pocket. These side-chain interactions become prominent during the transition of the Michaelis complex to the tetrahedral transition-state complex.

Amino Acid Sequence↗
PubMed1988

Structural and functional integrity of specificity and catalytic sites of trypsin.

The aspartic acid residue at the bottom of the substrate-binding pocket of trypsin was replaced by glutamic acid through site-directed mutagenesis. The wild-type (Asp-189) and mutant (Glu-189) trypsinogens were expressed in E. coli, purified to homogeneity, activated by enterokinase, and tested on a series of fluorogenic tetrapeptide substrates. The substrates were of the general formula succinyl-Ala-Ala-Pro-X-AMC, where AMC is 7-amino-4-methylcoumarin and X is Lys, Arg, or Orn (ornithine). As compared to Asp-189 trypsin, the activity of Glu-189 trypsin on lysyl and arginyl substrates decreased by 3-4 orders of magnitude while its Km values did not significantly change. Lengthening the side-chain of Asp-189 by one methylene group could not be compensated for by shortening the side-chain of the substrate, since Glu-189 trypsin had no measurable activity on the ornithyl substrate. The replacement of Asp-189 with glutamic acid at the base of the substrate-binding pocket of trypsin appears to distort the structure of the critical transition-state complex. This could happen by disrupting interactions normally associated with Asp-189, and by altering the relative position of the scissile peptide bond in the active site of the enzyme.

Aspartic Acid↗