Isolation and identification of thymosin α-1 from calf spleen using high performance liquid chromatography

A peptide isolated from calf spleen has been identified as thymosin α₁. The isolation involved defatting of the desiccated glands with acetone, extraction of the acetone power with pyridine acetate pH 5.5, heat denaturation, reverse phase chromatography on an RP-8 column, anion exchange chromatography on a Partisil SAX column and, finally, reverse phase purification on a μBondapak C₁₈ column. The identification was based on: 1) amino acid analysis; 2) thermolysin digest; and 3) retention time in two different HPLC systems. The amount isolated from the spleen was 10–20% of that isolated from calf thymus glands.     

Synthesis and biological evaluation of pNPY fragments

Peptide fragments of pNPY corresponding to the C'-terminal segments (13-36) and (25-36). the N-terminal segments (1-12) and (1-24), the segments (6-14) and (7-20), which contain a putative β-turn, and the internal segments (13-24) and (20-30) were synthesized using solid phase methodology. These fragments were assayed for NPY receptor binding activity in the rat hypothalamus membrane preparation, enhancement of food intake in the rat following ivt administration and inhibition of electrically stimulated muscle contraction in the rat vas deferens. Only the C-terminal fragment (13-36) retained some of the activities of pNPY, appearing to act as a weak agonist, having an additive effect with pNPY on the inhibition of muscle contraction and prolonging the duration of action of pNPY in the feeding assay. It also had considerable α-helical character, as did pNPY. None of the other peptide fragments had any agonist or antagonist activity. These results suggest that the expression of full biological NPY activity requires both the C- and the N-terminal segments as well as a putative amphiphilic α-helical segment (14-31).     

PREPARATIVE OXIDATIVE CONVERSION OF PROTECTED PEPTIDE Cα-HYDRAZIDES INTO THE CORRESPONDING ACIDS BY N-BROMOSUCCINIMIDE

A procedure for the preparative, oxidative conversion of protected peptide hydrazides into the corresponding acids by N-bromosuccinimide is described. Purification was achieved by high performance liquid chromatography on silica gel in chloroform-alcohol-acetic acid systems. Average recovery of purified protected peptide acids was over 90%.     

Structure activity of C-terminal modified analogs of Ac-CCK-7

Previous work indicates that both the C-terminal phenylalanine amide and the tryptophan moieties of chole-cystokinin (CCK) are critical pharmacophores for interaction with either the A or B receptor subtypes. We have examined a series of analogs of Ac-CCK-7 [Ac-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-Phe³³-NH₂] (2) in which the phenyl ring of the C-terminal Phe-NH₂ has been modified. Compounds were assessed in binding assays using homogenated rat pancreatic membranes and bovine striatum as the source of CCK-A and CCK-B receptors respectively and for anorectic activity after intraperitoneal administration to rats. Substitution of a number of cycloalkyi or bicyclic aryl moieties for the phenyl ring of phenylalanine³³ including cyclopentyl (20), cyclohexyl (21), cyclooctyl (23), 2-(5,6,7,8-tetrahydro)naphthyl (26), 2-naphthyl (27), and 1-naphthyl (29) led to analogs with 10–70 times the anorectic potency of 2. The anorectic activity of 21 was blocked by the specific CCK-A receptor antagonist MK-329. Other bulky aliphatic groups in place of the phenylalanine³³ aromatic ring such as isopropyl, 2-adamantyl and cyclohexylmethyl gave derivatives similar to 2 in potency. While most of the new compounds were comparable to CCK in binding assays, 23, 26, 27 and 29 were exceptionally potent with IC₅₀s 10^-11-10^-14 M in the pancreas. Compounds 23 and 29 were further evaluated for their ability to stimulate amylase secretion and found to have potencies similar to that of CCK. The dissociation between potency in the binding and amylase secretion assays suggests that they may interact with a high affinity binding site which is not coupled to amylase secretion. We conclude that CCK receptors possess a generous hydrophobic pocket capable of accommodating large alkyl groups in place of the side chain of phenylalanine³³ and that the pharmacological profile of CCK analogs can be tailored by appropriate exploitation of this finding.     

Applications of BOP reagent in solid phase peptide synthesis III. Solid phase peptide synthesis with unprotected aliphatic and aromatic hydroxyamino acids using BOP reagent

The BOP reagent [benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate], which has been shown to be ideally suited for solid phase synthesis, has now been found to be useful for solid phase synthesis using a minimal side-chain protection scheme. This new application of the BOP reagent was exemplified by the successful synthesis of the CCK-7 analog, Ac-Tyr(SO₃H)-Met-Gly-Trp-Met-Thr(SO₃H)-Phe-NH₂, using unprotected Boc(hydroxy)-amino acids [Boc-Thr-OH and Boc-Tyr-OH]. N-Terminal acetylation was achieved under mild conditions by using the BOP coupling reaction with acetic acid. This procedure provided the unprotected (Tyr²⁷, Thr³²)-peptide-resin which is ready for the required sulfation on the solid support without selective side-chain deprotection of Ty²⁷ and Thr³². Solid phase sulfation was evaluated under a variety of conditions and it was determined that disulfation was optimal using pyridine acetyl sulfate (38 equiv.) in pyridine at 45° for 4 h. Shorter reaction times or milder conditions lead to the formation of the Thr³² monosulfated analog. Cleavage of the disulfated analog from the PAM-resin was achieved using liquid ammonia and the product was purified by preparative hplc and fully characterized. The advantages of the new procedure are compared with the reported synthesis of CCK-7.     

TREATMENT OF ERECTILE DYSFUNCTION AFTER KIDNEY TRANSPLANTATION WITH INTRACAVERNOSAL SELF-INJECTION OF PROSTAGLANDIN E1

Purpose

We evaluate the results of treatment of erectile dysfunction in kidney transplant patients with intracavernosal self-injection of vasoactive drugs.

Materials and Methods

We evaluated and treated 26 male kidney transplant patients for erectile dysfunction. All patients had stable kidney function 6 to 75 months (mean 26.6 +/− 9) after transplantation. Each patient received an intracavernosal injection of 20 micro g. prostaglandin E1 (PGE1), and after 20 to 30 minutes the response was assessed. Nonresponders received 40 micro g. PGE1 at another visit, and those who showed no response were reinjected with 40 micro g. PGE1 plus 30 mg. papaverine hydrochloride. A total of 21 patients were enrolled in a self-injection program and have been followed between 3 and 21 months (mean 11.6 +/− 2.7).

Results

Hormonal alterations were seen in 7 patients with serum testosterone as low as 16.6 ng./ml. (normal 33 to 100), and testosterone infections gave only marginal response in 2. Intracavernosal injection of 20 micro g. PGE1 provided good response in 15 patients (57.7%), while 40 micro g. PGE1 alone or in combination with 30 mg. papaverine resulted in good response in another 6 and 2 patients, respectively. Among the 21 patients who were enrolled in the self-injection program 19 (90.5%) reported complete satisfaction with no adverse local or systemic complications except for local pain at the injection site in 4. There has been no change in serum creatinine, cyclosporine level or doses of immunosuppression medications during the observation period.

Conclusions

Intracavernosal self-injection of PGE1 is well accepted and tolerated by kidney transplant patients. It poses no apparent risks to the transplanted kidney and could be a good modality to treat erectile dysfunction in kidney transplant recipients.     

The Phenotype of a CASQ2 Mutation in a Saudi Family with Catecholaminergic Polymorphic Ventricular Tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) manifests with episodic syncope or sudden death in young patients following physical activity or emotional stress. The autosomal recessive form of CPVT is caused by mutations in the CASQ2 gene. In a consanguineous family, a novel homozygous CASQ2 mutation (p.L77P) was identified in a child with CPVT who required implantation of a cardioverter defibrillator due to episodes of syncope while on medical therapy. Genetic testing found the younger sibling, who had normal initial clinical screening, to be affected. Our cases underscore the importance of family screening through genetic testing to preemptively apply the appropriate medical intervention in CPVT. (PACE 2013; 36:e140–e142)     

Structure activity studies of tryptophan30 modified analogs of Ac-CCK-7

Cholecystokinin represents a family of gut hormones which among other activities, have been proposed to participate in satiety signaling. Ac-CCK-7 [Ac-Tyr(SO₃H)-Met-Gly-Trp³⁰-Met-Asp-Phe-NH₂ (2)] possesses the full spectrum of activity and potency of the intact hormone; thus analogs of 2 may be useful as anorectic agents. A series of derivatives has been prepared in which the tryptophan indole moiety of 2 has been modified. The new compounds were assayed in CCK binding assays using homogenated rat pancreatic membranes and bovine striatum as a source of CCK-A and CCK-B receptors respectively and in vivo in rats for anorectic activity. Although previous studies have concluded that the indole ring of Trp³⁰ is a critical pharmacophore for the interaction of CCK with both its A and B type receptors, we find 2-Nal³⁰-Ac-CCK-7 (20) to be nearly equipotent to 2 in both CCK binding and as an anorectic agent sensitive to blockade by the Merck CCK-A receptor antagonist MK-329. The extreme structural sensitivity of this anorectic activity is illustrated by the l-naphthylalanine³⁰ (19) and (benzo[b]thien-2-yl)alanine³⁰ (21) analogs which are 30 and 100 times less potent than 2 respectively. Other mono- and bicyclic Trp³⁰ replacements, including substituted phenylalanines, 3-quinolinylalanine, and 2-(5,6,7,8-tetrahydro)naphthylalanine, gave inactive compounds.     

Confirmation of the primary structure of thymosin α1 by microsequence analysis of limited acid and enzymatic hydrolysis fragments

The primary structure of the 28-peptide thymosin α₁ as determined by Goldstein et al. (1) has been confirmed by independent procedures. Limited dilute acid digestion generated a 26-peptide and a 22-peptide both extending to the C-terminal and lacking the N-terminal blocking group. A combination of Edman microsequencing, carboxypeptidase Y and thermolysin digestion, and fast atom bombardment mass spectrometry was used.