Peptide synthesis catalyzed by the Glu/Asp-specific endopeptidase. Influence of the ester leaving group of the acyl donor on yield and catalytic efficiency.

We recently described a two-step enzymatic semisynthesis of the superpotent analog of human growth hormone releasing factor, [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)-NH2 (4), from the precursor, [Ala15,29]-GRF(4-29)-OH (1). C-Terminal amidation of 1 to form [Ala15]-GRF(4-29)-NH2 (2) was achieved by carboxypeptidase-Y-catalyzed exchange of Ala29-OH for Arg-NH2. The target analog 4 was then obtained by acylation of segment 2 with desNH2Tyr-D-Ala-Asp(OH)-OR (3) (R = CH3CH2- or 4-NO2C6H4CH2-) catalyzed by the V8 protease.

Solid-phase synthesis of extended lactam ring systems: preparation of amino acid alpha-fluorenylmethyl esters for the synthesis of reverse-extended lactams.

Conventional side-chain to side-chain cyclization between Lys/Orn and Asp/Glu side chains has the inherent shortcoming of limited variability in the ring sizes; limited by the amino acids involved in the lactamization. A novel approach to modulate the ring size has been introduced by using "spacers" (e.g.

Radioimmunoassays for the C-terminus of prothymosin alpha and the N-terminus of parathymosin alpha for the measurement of the levels of alpha-thymosins in human cancer.

A radioimmunoassay specific for the C-terminus of human prothymosin alpha was developed using the synthetic peptide [Cys-Aca degrees]-human prothymosin alpha (90-109)-OH coupled to KLH as antigen and the analogue [Tyr-Aca degrees]-human prothymosin alpha (90-109)-OH labelled with 125I as tracer. The radioimmunoassay measured intact prothymosin alpha, in the range of 2-100 pmol and does not cross-react with the partly homologous polypeptide parathymosin alpha. A major epitope was located in the segment 95-107.

Risk factors for homelessness among schizophrenic men: a case-control study.

Objectives: To identify risk factors for homelessness among the severely mentally ill, we conducted a case-control study of 100 indigent schizophrenic men meeting criteria for literal homelessness and 100 such men with no homeless history.

Methods: Subjects were recruited from shelter, clinic, and inpatient psychiatric programs in Upper Manhattan. Clinical interviewers employed standardized research instruments to probe three domains of risk factors: severity of mental illness, family background, and prior mental health service use.

Pegylated peptides. II. Solid-phase synthesis of amino-, carboxy- and side-chain pegylated peptides.

General procedures are presented for the site-specific pegylation of peptides at the NH2-terminus, side-chain positions (Lys or Asp/Glu) or COOH-terminus using solid-phase Fmoc/tBu methodologies. A model tridecapeptide fragment of interleukin-2, IL-2(44-56)-NH2, was chosen for this study since it possesses several trifunctional amino acids which serve as potential sites for pegylation. The pegylation reagents were designed to contain either Nle or Orn, which served as diagnostic amino acids for confirming the presence of 1 PEG unit per mole of peptide.

Enhanced stability and potency of novel growth hormone-releasing factor (GRF) analogues derived from rodent and human GRF sequences.

Native human GRF(1-44)-NH2(hGRF44) is subject to biological inactivation by both enzymatic and chemical routes. In plasma, hGRF44 is rapidly degraded via dipeptidylpeptidase IV (DPP-IV) cleavage between residues Ala2 and Asp3. The hGRF44 is also subject to chemical rearrangement (Asn8-->Asp8, beta-Asp8 via aminosuccinimide formation) and oxidation [Met27-->Met(O)27] in aqueous environments, greatly reducing its bioactivity.

Follow-up of chronically homeless mentally ill men.

Objective: To supply information on the efficacy of on-site day treatment for homeless mentally ill men in shelters, the authors followed up homeless mentally ill men 18 months after placement in community housing.

Method: The 42 subjects had been evaluated before and 6 months after entering an on-site day treatment program. The authors reinterviewed 34 of these patients again 1 year after the first follow-up to determine housing status, hospitalization, aftercare, criminal justice contacts, income, and employment.

Characterization of the rat neutral and basic amino acid transporter utilizing anti-peptide antibodies.

High-titer, site-specific antibodies have been produced against the rat kidney broad-spectrum, sodium-independent neutral and basic amino acid transporter (NBAA-Tr) whose cDNA we cloned earlier. These antibodies have allowed us to characterize the transporter protein in normal rat tissues and in various cellular and in vitro expression systems. Western analysis detected 84- to 87-kDa glycosylated species enriched in rat renal and jejunal epithelial cell brush border membranes.

Pegylated peptides I: Solid-phase synthesis of N alpha-pegylated peptides using Fmoc strategy.

The feasibility of pegylating peptides by the solid-phase procedure was examined. Although polyethyleneglycol (PEG) was shown to be partially degraded by HF, the use of TFA was fully compatible with the PEG system. Therefore, the Fmoc/tBu solid-phase strategy was utilized for the synthesis of a series of model tetra-, octa- and dodecapeptides, and the corresponding N alpha-pegylated peptides, which were prepared from common peptide-resin intermediates.

Oncocytic tumors of salivary gland type: a study with emphasis on nuclear DNA ploidy.

We studied nine cases of oncocytic tumors of salivary gland type in order to evaluate their clinico-pathologic profiles and nuclear DNA patterns as criteria for the differential diagnosis between benign and malignant forms. The tumors were located at the parotid gland, palate, and orbit. The age of the patients ranged between 35 and 85 years and the sex ratio (F:M) was 1:0.

Enzymatic semisynthesis of a superpotent analog of human growth hormone-releasing factor.

A superpotent analog of human growth hormone-releasing factor, [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)-NH2 (4), was prepared from the precursor, [Ala15,29]-GRF(4-29)-OH (1), by a two-step enzymatic semisynthesis. The amidated C-terminus, essential for high biological potency, was obtained via a carboxypeptidase Y-catalyzed exchange of Ala29-OH for Arg29-NH2 to produce [Ala15]-GRF(4-29)-NH2 (2). The N-terminal desNH2Tyr-D-Ala moiety, which greatly increases in vivo duration of action, was then incorporated by V8 protease-catalyzed condensation of segment 2 with desNH2Tyr-D-Ala-Asp(OH)-OR [R = CH3CH2- (3a) or 4-NO2C6H4CH2-(3b)].

Semisynthesis of human growth hormone-releasing factor by trypsin catalyzed coupling of leucine amide to a C-terminal acid precursor.

Human growth hormone-releasing factor, GRF(1-44)-NH2, was synthesized by trypsin catalyzed coupling of Leu-NH2 to Arg43 of the precursor, GRF(1-43)-OH, prepared by solid phase peptide synthesis. The semisynthetic GRF(1-44)-NH2 was fully characterized and showed full potency in the rat pituitary in vitro bioassay. Conversion to GRF(1-44)-NH2 was limited to 60-70% in both 75% v:v N,N'-dimethylacetamide and 95% v:v 1,4-butanediol due to competing transpeptidations at Arg41 and Arg38 generating [Leu42]-GRF(1-42)-NH2 and [Leu39]-GRF(1-39)-NH2 side-products, respectively.

Synthesis and biological activity of novel C-terminal-extended and biotinylated growth hormone-releasing factor (GRF) analogs.

A series of novel hGRF(1-29)-NH2 analogs were synthesized and biotinylated. The immunological and biological activities of these analogs were then characterized. To distance the biotin moiety from the putative bioactive core, a C-terminal spacer arm consisting of -Gly-Gly-Cys-NH2 (-GGC) was added to hGRF(1-29)-NH2 (hGRF29) and analogs, with subsequent biotinylation performed at the cysteine residue.

Semisynthesis of human growth hormone-releasing factors by alpha-amidating enzyme catalyzed oxidation of glycine-extended precursors.

Recombinant alpha-amidating enzyme was used in the semisynthesis (1-5 mg scale) of human growth hormone-releasing factor, GRF(1-44)-NH2, by in vitro enzymatic oxidation of the glycine-extended precursor, GRF(1-44)-Gly-OH, prepared by solid-phase synthesis. The equipotent analog, GRF(1-29)-NH2, and the superactive analog, [Ala15]-GRF(1-29)-NH2, were also prepared by this route and were fully characterized. Isolated yields of about 75% were obtained, and the products each possessed full potency in an in vitro rat pituitary bioassay and full receptor-binding affinity.

Solution structures of cyclic and dicyclic analogues of growth hormone releasing factor as determined by two-dimensional NMR and CD spectroscopies and constrained molecular dynamics.

Solution structures were determined for a linear analogue of growth hormone releasing factor (GRF), and cyclic and dicyclic analogues in which the side chains of aspartyl and lysyl residues spaced at positions i-(i + 4) were joined to form a lactam. The four analogues were [Ala15]-GRF-(1-29)-NH2 and its cyclo8-12, cyclo21-25, and dicyclo8-12;21-25 derivatives. The peptides were studied in two solvent systems: 75% methanol/25% water at pH 6.

A sensitive and specific competition microELISA for the immunoactive polypeptide parathymosin and detection of this peptide in porcine tissues.

A sensitive and specific microELISA assay is described for the immunoactive polypeptide parathymosin. Antibodies against a synthetic peptide corresponding to the rat parathymosin sequence 5-30 were raised in rabbits immunised with this peptide conjugated to keyhole limpet hemocyanin (KLH). The useful range of the assay was 0.

Degradation of aspartic acid and asparagine residues in human growth hormone-releasing factor.

Products of the degradation of human growth hormone-releasing factor (GRF) in aqueous solutions (15-200 microM) have been isolated and fully characterized. The cleavage product, GRF(4-44)-NH2, and the isomerization product, [beta-Asp3]GRF(1-44)-NH2, from the degradation of GRF(1-44)-NH2 in acidic solution and the corresponding products, GRF(4-29)-NH2 and [beta-Asp3]GRF(1-29)-NH2, from the degradation of GRF(1-29)-NH2 have been isolated and characterized. The products, [beta-Asp8]GRF(1-44)-NH2 and [Asp8]GRF(1-44)-NH2, from the deamidation of GRF(1-44)-NH2 at pH 8.

Biosynthesis of human growth hormone-releasing hormone (hGRH) in the pituitary of hGRH transgenic mice.

We have studied the posttranslational processing of prohuman GH-releasing hormone (pro-hGRH) to the mature hormones, hGRH(1-44)-NH2 and hGRH(1-40)-OH, and its carboxyl-terminal peptide (hGCTP) in pituitary cells from transgenic mice bearing a metallothionein-hGRH fusion gene after incubation with [35S]methionine. After separation on HPLC, 35S-labeled and unlabeled hGRH in medium and cell extracts were characterized by RIA and immunoprecipitation with antisera against hGRH and against hGCTP. After a 4-h pulse, unlabeled and [35S]pro-hGRH, hGRH(1-44)-NH2, and hGRH(1-40)-OH were identified in medium and cell extracts by both RIA and immunoprecipitation with anti-hGRH serum.

Effect of human growth hormone-releasing factor and a potent analog on antibody formation in African green monkeys.

African Green monkeys were injected (2 x daily subcutaneously for six months) with human GRF(1-44)-NH2 (10 micrograms/kg BW) or a more potent analog, [desNH2Tyr1,Ala15]-hGRF(1-29)-NH2 (2 micrograms/kg BW) to determine the potential of each peptide to induce antibody formation. Blood samples were taken every two weeks, diluted 1:100 and tested for ability to bind radioiodinated hGRF. One animal in the hGRF(1-44)-NH2 group [N = 6] produced low-titer GRF antibodies by 6 weeks (19% binding) and continued throughout the 24 weeks of treatment (average = 50-60% binding).

Type-specific and cross-reactive epitopes in human papillomavirus type 16 capsid proteins.

Genital human papillomavirus (HPV) 16 infection is frequently associated with cancer of the uterine cervix, as well as with precancerous lesions. In order to generate serologic reagents which might be useful in the diagnosis of HPV 16 infection, rabbit polyclonal and mouse monoclonal antisera were raised to carboxy terminal peptides from the HPV 16 L1 and L2 open reading frames (ORFs). Anti-L1 and -L2 peptide sera recognized HPV 16 L1 and L2 fusion proteins in Western blots and by immunoprecipitation.

Synthesis and biological evaluation of mouse growth hormone-releasing factor.

The recently described mouse growth hormone-releasing factor (mGRF) was synthesized by the solid phase procedure, purified by 2 stages of preparative high performance liquid chromatography and fully characterized. The biologic activity of the 42-amino acid peptide (H-His-Val-Asp-Ala-Ile-Phe- Thr-Thr-Asn-Tyr- Arg-Lys-Leu-Leu-Ser-Gln-Leu-Tyr-Ala-Arg-Lys-Val-Ile-Gln-Asp-Ile-Met-Asn- Lys- Gln-Gly-Glu-Arg-Ile- Gln-Glu-Gln-Arg-Ala-Arg-Leu-Ser-OH) was assessed in primary cultures of both mouse and rat anterior pituitary cells and compared to synthetic rat (rGRF) and human (hGRF) growth hormone-releasing factors. mGRF was equipotent to rGRF in mouse somatotrophs but slightly less potent in rat somatotrophs, while hGRF was 3-5 times less potent in both rodent species.

GRF analogs and fragments: correlation between receptor binding, activity and structure.

GH-releasing activity in vitro was directly correlated with GRF receptor binding affinity for all hGRF analogs examined. hGRF(1-29)-NH2 analogs with Ala15-substitution (for Gly15) displayed 4-5 times higher affinity for the GRF receptor relative to hGRF(1-44)-NH2. Replacement of Gly15 with Sar15 resulted in a dramatic loss of activity and receptor binding.

Identification of 11,13-dihydroxy-14-octadecaenoic acid as a circulating Na+/K(+)-ATPase inhibitor.

Two digitalis-like compounds (DLC) were purified to homogeneity from bovine plasma. The purification procedure consisted of organic extractions and batch chromatography followed by three subsequent separations using reverse-phase high-performance liquid chromatography. The presence of a DLC in the different fractions was monitored by their ability to inhibit (a) [3H]ouabain binding to rat brain synaptosomes, and (b) microsomal Na+/K(+)-ATPase activity.

In vitro stability of growth hormone releasing factor (GRF) analogs in porcine plasma.

The stability of growth-hormone releasing factor (growth regulating factor; GRF) analogs in porcine plasma was examined. GRF analogs were incubated in porcine plasma at 37 degrees C, extracted and subsequently analyzed using high performance liquid chromatography (HPLC). GRF(1-29)-NH2 was rapidly broken down in the plasma with a degradation rate of t1/2 = 13 min.

Comparison of enzymatic semisyntheses of peptide amides: human growth hormone releasing factor and analogs.

Enzymatic semisyntheses of growth hormone releasing factor (GRF), a 44-residue peptide amide hormone, from C-terminal acid precursors, are compared. A recombinant alpha-amidating enzyme was used to convert the glycine-extended precursor, GRF(1-44)-Gly-OH, to GRF(1-44)-NH2 in an essentially quantitative fashion. Trypsin was used to convert the precursors, GRF(1-43)-OH and GRF(1-44)-OH, to GRF(1-44)-NH2 (60 and 15% conversion, respectively) in a 75% v:v N,N'-dimethylacetamide solution containing a large excess of leucine amide.